Use of halogenase enzymes in the catalytic synthesis of 2'-halogenated nucleosides

Abstract

The application discloses application of a halogenase in catalyzing synthesis of 2'-halogenated nucleosides. The halogenase is any one of NSH1, NSH2, NSH3, NSH4 and NSH5, and the substrate used in the catalysis is any one of 2'-deoxyguanosine, 2'-deoxyinosine and 2'-deoxyadenosine. The biosynthesis method has the advantages of high stereoselectivity, high product yield, simple halogen source, easy control of operation process, low production cost, green and environmentally-friendly reaction and high catalytic activity, and provides a new method for synthesis of 2'-halogenated compounds.

Description

Technical Field

[0001] This application relates to the field of biosynthesis technology, and in particular to the application of halogenases in the catalytic synthesis of 2'-halogenated nucleosides. Background Technology

[0002] Nucleoside analogs are an important class of drugs with activity against a variety of biological targets. Modifications at different positions of the ribose and bases of a nucleoside analog result in different pharmacological properties. Modification at the 2' ribose site is a particularly important type of nucleoside modification, used in both nucleoside analog drugs and the nucleoside building blocks of therapeutic oligonucleotides.

[0003] For example, 2'-methoxy, 2'-methoxyethoxy (MOE), and 2'-fluoro-modified nucleosides are common substituents in therapeutic oligonucleotides. These modifications significantly enhance nuclease resistance and increase RNA binding affinity. However, the synthesis of 2'-modified nucleosides typically requires multiple steps and involves extensive protection / deprotection steps, and stereoselectivity control is difficult, making the synthesis of these molecules challenging.

[0004] 2'-Halogenated nucleosides are compounds in which a halogen (such as fluorine, chlorine, bromine, or iodine) is introduced onto the 2'-carbon atom of a nucleoside molecule. As an important nucleoside derivative, 2'-halogenated nucleosides have wide applications in the fields of medicine and biotechnology. The synthesis of 2'-halogenated nucleosides also faces the following problems: (1) Nucleoside molecules have complex structures, especially with multiple active sites on the deoxyribose ring that may react. In the chemical synthesis process, achieving highly selective halogenation of the 2'-site is a challenge, often requiring expensive reagents and complex protection-deprotection strategies, which increases the number of reaction steps and costs. (2) The chemical synthesis of 2'-halogenated nucleosides usually requires strong acid, strong base, or high temperature conditions. These conditions may cause the glycosidic bond of the nucleoside to break or the base to degrade, thereby reducing the yield and purity of the target product. In addition, the introduction of different halogens (such as fluorine, chlorine, and bromine) requires different reaction conditions, further increasing the difficulty of process optimization. Although biocatalysis has gained popularity in recent years, and the synthesis of 2'-halonucleotides using halogenases offers advantages such as mild reaction conditions and high selectivity, current halogenases still have limitations in terms of substrate adaptability, activity, and yield. Therefore, developing novel halogenases to achieve efficient and specific 2'-halonucleotide synthesis remains a significant technical challenge. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this application proposes a rapid and simple biocatalytic method for achieving highly stereoselective modification of the C2' site of 2'-deoxynucleoside substrates.

[0006] This invention discloses the application of halogenases in the catalytic synthesis of 2'-halonucleotides, wherein the halogenase is any one of NSH1, NSH2, NSH3, NSH4, and NSH5, the amino acid sequence of NSH1 is shown in SEQ ID NO.1, the amino acid sequence of NSH2 is shown in SEQ ID NO.2, the amino acid sequence of NSH3 is shown in SEQ ID NO.3, the amino acid sequence of NSH4 is shown in SEQ ID NO.4, and the amino acid sequence of NSH5 is shown in SEQ ID NO.5;

[0007] The substrate used in the catalysis is any one of 2'-deoxyguanosine, 2'-deoxyinosine, and 2'-deoxyadenosine.

[0008] 2'-Halogenated nucleosides are an important class of pharmaceutical intermediates, widely used in the synthesis of antiviral and anticancer drugs.

[0009] The 2'-deoxyguanosine, 2'-deoxyinosine, and 2'-deoxyadenosine used in this invention belong to the 2'-deoxynucleosides, which are composed of ribose and bases, do not contain phosphate groups, and are not chemically affected by phosphate groups, thus achieving specific C2'-position halogenation on substrates (2'-deoxynucleosides) that do not contain phosphate groups.

[0010] In some implementations, expressed and purified halogenases NSH1, NSH2, NSH3, NSH4, or NSH5 are used as biocatalysts to catalyze the halogenation of nucleoside substrates.

[0011] In some embodiments, the catalytic reaction system comprises: any one of halogenases NSH1, NSH2, NSH3, NSH4, and NSH5, ferrous ions, L-ascorbic acid, α-ketoglutarate, a halide salt, and the substrate.

[0012] In some embodiments, the halide is a chloride salt or a bromide salt.

[0013] In some embodiments, the chloride salt is sodium chloride or potassium chloride.

[0014] In some embodiments, the bromide salt is sodium bromide or potassium bromide.

[0015] In some embodiments, the reaction solution is a buffer solution with a pH of 6 to 8, and the reaction temperature is 20 to 40°C.

[0016] In some embodiments, the expression step includes: constructing the gene sequence of the halogenase into an expression vector to obtain a recombinant vector, and transforming it into Escherichia coli to induce the expression of the halogenase protein.

[0017] In some embodiments, the purification is performed by purifying the halogenated protein using a nickel column.

[0018] In summary, compared with the prior art, this application achieves the following technical effects:

[0019] (1) This application discloses five Fe(II) / α-ketoglutarate-dependent halogenases NSH1, NSH2, NSH3, NSH4 and NSH5. Any one of these enzymes can selectively halogenate nucleoside substrates and catalyze the halogenation of 2'-deoxynucleosides (2'-deoxyguanosine, 2'-deoxyinosine, 2'-deoxyadenosine) to directly synthesize 2'-halogenated products in nucleoside form, providing a new method for the development of related drugs.

[0020] (2) The biosynthesis method of the halogenated nucleotides of this application has high stereoselectivity, high chiral selectivity for chlorination at the C2' site, and halogen is added to the same side of the C3' hydroxyl group.

[0021] (3) The biosynthesis method of halogenated nucleotides in this application has high conversion rate, simple halogen source, easy operation process control, low production cost, green and environmentally friendly reaction and high catalytic activity. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The results are liquid chromatography results of the chlorination reaction of 2'-deoxyadenosine substrate catalyzed by Fe(II) / α-ketoglutarate-dependent halogenases NSH1, NSH2, NSH3, NSH4 and NSH5 in Example 5.

[0024] Figure 2 Liquid chromatography-mass spectrometry analysis of 2'-chloro-deoxyadenosine, the chlorination product of Example 5.

[0025] Figure 3 The 1H NMR spectrum analysis of 2'-chloro-deoxyadenosine, the chlorination product of Example 5.

[0026] Figure 4 The results are liquid chromatography results of the chlorination reaction of the 2'-deoxyguanosine substrate catalyzed by Fe(II) / α-ketoglutarate-dependent halogenases NSH1, NSH2, NSH3, NSH4 and NSH5 in Example 6.

[0027] Figure 5 Liquid chromatography-mass spectrometry analysis of 2'-chloro-deoxyguanosine, the chlorination reaction product of Example 6.

[0028] Figure 6 The 1H NMR spectrum analysis of 2'-chloro-deoxyguanosine, the chlorination reaction product of Example 6.

[0029] Figure 7 The results are liquid chromatography results of the chlorination reaction of 2'-deoxyinosine substrate catalyzed by Fe(II) / α-ketoglutarate-dependent halogenases NSH1, NSH2, NSH3, NSH4 and NSH5 in Example 7.

[0030] Figure 8 Liquid chromatography-mass spectrometry analysis of 2'-chloro-deoxyinosine, the chlorination reaction product of Example 7.

[0031] Figure 9 The liquid chromatography results are for the halogenase-catalyzed bromination reaction of 2'-deoxyadenosine substrate in Example 8.

[0032] Figure 10 Liquid chromatography-mass spectrometry analysis of 2'-bromo-deoxyadenosine, the bromination product of Example 8.

[0033] Figure 11 The liquid chromatography results are for the halogenase-catalyzed bromination reaction of the 2'-deoxyguanosine substrate in Example 9.

[0034] Figure 12 Liquid chromatography-mass spectrometry analysis of 2'-bromo-deoxyguanosine, the bromination product of Example 9. Detailed Implementation

[0035] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present application.

[0036] The objective of this application can be achieved through the following technical solutions:

[0037] The gene sequences of halogenase NSH1 (derived from sea bacterium flounder), NSH2 (derived from marine bacterium burvini), NSH3 (derived from terrestrial myxobacteria), and NSH4 and NSH5 (derived from bacteria of the order Bdellovibrio) were introduced into *Escherichia coli* to induce the expression of halogenases NSH1, NSH2, NSH3, NSH4, or NSH5 proteins. The target proteins were then purified by nickel column chromatography. The purified Fe(II) / α-ketoglutarate-dependent halogenases can catalyze the selective chlorination of nucleoside substrates 2'-deoxyguanosine (2'-dG), 2'-deoxyinosine (2'-dI), and 2'-deoxyadenosine (2'-dA) at the C2' site.

[0038] Specifically, the following steps are included:

[0039] (1) The halogenase NSH1-5 gene fragments derived from *Botrytis cinerea*, *Botrytis cinerea*, terrestrial myxobacteria, and *Bdellovibrio* were synthesized separately, and the target gene was ligated into a plasmid vector to obtain a recombinant expression plasmid; the plasmid vector was pET28a, and the restriction endonucleases used to ligate the target gene fragment into the plasmid vector were NdeI and XholI; the inducer was isopropyl-β-D-thiogalactoside (IPTG).

[0040] Constructing recombinant expression of Fe(II) / α-ketoglutarate-dependent halogenase-producing bacteria derived from light yellow sea myxobacteria, marine damselfly bacterium, terrestrial myxobacteria, and bacteria from the order Bdellovibrioles: The recombinant expression plasmid obtained in step (1) was introduced into Escherichia coli BL21(DE3) to obtain the desired engineered bacteria for recombinant expression.

[0041] (2) Expression of Fe(II) / α-ketoglutarate-dependent halogenases derived from light yellow sea myxobacteria, marine damselfly bacterium, terrestrial myxobacteria and bacteria from the order Bdellovibrio: The engineered bacteria obtained in step (2) were cultured in liquid culture medium (20~80 μg / mL kanamycin) for scale-up. When the engineered bacteria grew to the logarithmic growth phase, the temperature was lowered and an inducer was added to induce the expression of halogenases. After 16 hours, engineered bacteria with the target protein were obtained.

[0042] (3) Protein purification of Fe(II) / α-ketoglutarate-dependent halogenases derived from light yellow sea myxobacteria, marine dammbovesporium, terrestrial myxobacteria and bacteria from the order Bdellovibrioles: The engineered bacteria obtained in step (3) were crushed to obtain crude enzyme solution, and the target protein was purified by nickel column to obtain a target protein with high purity.

[0043] (4) Halogenation of nucleosides: The protein obtained in step (3) is used as a biocatalyst to catalyze the halogenation of the nucleoside substrate. The reaction system is as follows: halogenase protein, ferrous ions, L-ascorbic acid, α-ketoglutaric acid, halide salts (sodium chloride / potassium chloride, sodium bromide / potassium bromide, etc.), and nucleoside substrate. The reaction solution is a buffer solution with a pH of 6-8, and the reaction temperature is room temperature. Halogenated nucleoside products can be generated within 12 hours.

[0044] This application utilizes *E. coli* and the pET28a plasmid vector, with NdeI and XholI as restriction endonucleases, and IPTG as the inducer. This system is easy to operate, mature, and stable, making the proposed method readily industrializable.

[0045] Example 1: Expression and purification of Fe(II) / α-ketoglutarate-dependent halogenase NSH1 derived from *Scutellaria baicalensis*.

[0046] The Fe(II) / α-ketoglutarate-dependent halogenase NSH1, derived from *Saccharomyces cerevisiae*, was synthesized using NdeI and XholI restriction endonucleases and constructed into a pET series vector. The amino acid sequence of NSH1 is shown in SEQ ID NO. 1. The vector containing the gene encoding the Fe(II) / α-ketoglutarate-dependent halogenase was transformed into *Escherichia coli* BL21(DE3) expression strain. The correctly sequenced expression strain was inoculated into LB liquid medium (10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl) containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm until OD500. 600 After inducing the bacterial culture to 0.8, the temperature was lowered to 16℃, and IPTG was added to a final concentration of 0.2 mM. After induction for 16 h, the bacterial cells were collected by centrifugation at 4000 rpm for 20 min. The bacterial cells were then purified using a nickel column to obtain the target protein in high purity.

[0047] Example 2: Expression and purification of Fe(II) / α-ketoglutarate-dependent halogenase NSH2 from Bovesella damm (Marine bromoxynil)

[0048] Using the same method as in Example 1, the Fe(II) / α-ketoglutarate-dependent halogenase NSH2 derived from *Bovrilium damm* was successfully expressed and purified. The amino acid sequence of NSH2 is SEQ ID NO.2.

[0049] Example 3 Expression and purification of Fe(II) / α-ketoglutarate-dependent halogenase NSH3 from terrestrial myxobacteria

[0050] Using the same method as in Example 1, the Fe(II) / α-ketoglutarate-dependent halogenase NSH3 derived from terrestrial myxobacteria was successfully expressed and purified. The amino acid sequence of NSH3 is SEQ ID NO.3.

[0051] Example 4: Expression and purification of Fe(II) / α-ketoglutarate-dependent halogenase NSH4 from Bdellovibrioles.

[0052] Using the same method as in Example 1, the Fe(II) / α-ketoglutarate-dependent halogenase NSH4 derived from Bdellovibrioles was successfully expressed and purified. The amino acid sequence of NSH4 is SEQ ID NO.4.

[0053] Example 5: Expression and purification of Fe(II) / α-ketoglutarate-dependent halogenase NSH5 from Bdellovibrioles.

[0054] Using the same method as in Example 1, the Fe(II) / α-ketoglutarate-dependent halogenase NSH5 derived from Bdellovibrioles was successfully expressed and purified. The amino acid sequence of NSH5 is SEQ ID NO. 5.

[0055] Example 6: Chlorination reaction of 2'-deoxyadenosine substrate by Fe(II) / α-ketoglutarate-dependent halogenases NSH1, NSH2, NSH3, NSH4 and NSH5

[0056] The halogenation reaction system of Fe(II) / α-ketoglutarate-dependent halogenase on 2'-deoxyadenosine substrate prepared using Examples 1-5 is as follows: 10 mM NaCl, 2 mM substrate, 3 mM α-ketoglutarate, 0.5 mM ferrous ions, 1 mM L-ascorbic acid, and an appropriate amount of purified enzyme (NSH1, NSH2, NSH3, NSH4, or NSH5) are added to HEPES buffer at pH 6-8.

[0057] The liquid chromatography results of the Fe(II) / α-ketoglutarate-dependent halogenase NSH1-5 catalyzing the 2'-deoxyadenosine monochlorination reaction are as follows: Figure 1 As shown, the conversion rate of NSH1 was approximately 100%, and 2'-deoxyadenosine was successfully converted to 2'-chloroadenosine. The conversion rates of NSH2 and NSH3 were approximately 50%, NSH4 and NSH5 were approximately 10%, and the conversion rates were approximately 5%. Liquid chromatography-mass spectrometry analysis of the reaction products confirmed that the products were chlorinated products. Figure 2 As shown, the halogenases in Examples 1-5 can catalyze the chlorination reaction of 2'-deoxyguanosine substrates.

[0058] The product was further purified and analyzed by proton nuclear magnetic resonance spectroscopy. The results are as follows: Figure 3As shown, by comparing the measured spectrum with the reported spectrum, it was confirmed that the chlorination position is C2' and the product is 2'-chloro-adenosine.

[0059] Example 7: Chlorination of 2'-deoxyguanosine catalyzed by Fe(II) / α-ketoglutarate-dependent halogenases NSH1, NSH2, NSH3, NSH4 and NSH5

[0060] The difference from Example 6 is that the substrate for the chlorination reaction in Example 7 is 2'-deoxyguanosine, and the catalyst is the halogenases NSH1, NSH2, NSH3, NSH4, or NSH5 purified in Examples 1-5. The liquid chromatography results of the substrate and product of the chlorination reaction of 2'-deoxyguanosine catalyzed by halogenases NSH1-5 are as follows: Figure 4 As shown, according to Figure 4 The substrate peak area is substituted into the substrate concentration standard curve to calculate the product concentration, and the substrate conversion rate is calculated. This is a commonly used quantitative method, and the conversion rate is approximately 5-95%.

[0061] Liquid chromatography-mass spectrometry analysis of the products of the chlorination reaction in Example 7 is as follows: Figure 5 As shown, the product was confirmed to be a chlorinated product, indicating that the halogenases in Examples 1-5 can all catalyze the chlorination reaction of the 2'-deoxyguanosine substrate. Further purification yielded a pure product, which was then analyzed by 1H NMR spectroscopy. The results are shown below. Figure 6 As shown, by comparing the measured spectrum with the reported spectrum, it was confirmed that the chlorination position is C2' and the product is 2'-chloro-deoxyguanosine.

[0062] Example 8: Chlorination of 2'-deoxyinosine catalyzed by Fe(II) / α-ketoglutarate-dependent halogenases NSH1, NSH2, NSH3, NSH4 and NSH5

[0063] The difference from Example 6 is that the substrate for the chlorination reaction in Example 8 was 2'-deoxyinosine, and the catalyst was the halogenases NSH1, NSH2, NSH3, NSH4, or NSH5 purified in Examples 1-5. The liquid chromatography results of the substrate and product of the chlorination reaction of 2'-deoxyinosine catalyzed by halogenases NSH1-5 are as follows... Figure 7 As shown, according to Figure 7 The substrate peak area is substituted into the substrate concentration standard curve to calculate the product concentration, and the substrate conversion rate is calculated. This is a commonly used quantitative method, and the conversion rate is approximately 5-95%.

[0064] Liquid chromatography-mass spectrometry analysis of the products of the chlorination reaction in Example 8 is as follows: Figure 8 As shown, the product was determined to be a chlorinated product, indicating that the halogenases in Examples 1-5 can all catalyze the chlorination reaction of the 2'-deoxyinosine substrate.

[0065] Example 9: Bromination of 2'-deoxyadenosine substrates by Fe(II) / α-ketoglutarate-dependent halogenases NSH1 and NSH2

[0066] The difference from Example 6 is that the halogen source in the reaction system of Example 9 is sodium bromide, and the catalyst is the halogenase NSH1 or NSH2 purified in Examples 1-2. The liquid chromatography results of the substrate and product of the 2'-deoxyadenosine bromination reaction catalyzed by halogenases NSH1 and NSH2 are as follows: Figure 9 As shown, according to Figure 9 The substrate peak area was substituted into the substrate concentration standard curve to calculate the product concentration, and the substrate conversion rate was calculated. This is a commonly used quantitative method. The results showed that the conversion rate of NSH1 was about 95% and the conversion rate of NSH2 was about 10%.

[0067] Liquid chromatography-mass spectrometry analysis of the products of the bromination reaction in Example 9 is as follows: Figure 10 As shown, the product was determined to be a brominated product, indicating that the halogenases in Examples 1 and 2 can both catalyze the bromination reaction of the 2'-deoxyadenosine substrate.

[0068] Example 10: Bromination of 2'-deoxyguanosine substrates by Fe(II) / α-ketoglutarate-dependent halogenases NSH3, NSH4, and NSH5

[0069] The difference from Example 7 is that the halogen source in the reaction system of Example 10 is sodium bromide, and the catalyst is the halogenase NSH3, NSH4, or NSH5 purified in Examples 3-5. The liquid chromatography results of the substrate and product of the 2'-deoxyguanosine bromination reaction catalyzed by halogenases NSH3, NSH4, and NSH5 are as follows: Figure 11 As shown, according to Figure 11 The substrate peak area was substituted into the substrate concentration standard curve to calculate the product concentration, and the substrate conversion rate was calculated. This is a commonly used quantitative method. The results showed that the conversion rate of NSH3 was about 40%, and the conversion rates of NSH4 and NSH5 were about 10%.

[0070] Liquid chromatography-mass spectrometry analysis of the products of the bromination reaction in Example 10 is as follows: Figure 12 As shown, the product was determined to be a brominated product, indicating that the halogenases in Examples 3, 4 and 5 can all catalyze the bromination reaction of the 2'-deoxyguanosine substrate.

[0071] Based on the above embodiments, this application discloses a method for the biosynthesis of 2'-halogenated nucleosides. The synthesis method is catalyzed by halogenases NSH1, NSH2, NSH3, NSH4 and NSH5. The amino acid sequence of NSH1 is shown in SEQ ID NO.1, the amino acid sequence of NSH2 is shown in SEQ ID NO.2, the amino acid sequence of NSH3 is shown in SEQ ID NO.3, the amino acid sequence of NSH4 is shown in SEQ ID NO.4, and the amino acid sequence of NSH5 is shown in SEQ ID NO.5.

[0072] This application identifies five Fe(II) / α-ketoglutarate-dependent halogenases, NSH1, NSH2, NSH3, NSH4, and NSH5, which can catalyze the halogenation of 2'-deoxynucleosides. The gene sequences of halogenases NSH1-5, derived from *Bovella simonii* (a species of myxobacterium in the Yellow Sea), *Bovella damm* (a species of marine myxobacterium), terrestrial myxobacteria, and *Bdellovibrio* (a species of bacteria), were introduced into *Escherichia coli*. This induced the expression of the halogenases NSH1, NSH2, NSH3, NSH4, or NSH5 proteins in *E. coli*. The target proteins were then purified using a nickel column. The purified Fe(II) / α-ketoglutarate-dependent halogenases can catalyze the selective halogenation of nucleoside substrates 2'-deoxyguanosine (2'-dG), 2'-deoxyinosine (2'-dI), and 2'-deoxyadenosine (2'-dA) at the C2' site. The biosynthesis method of this application has the advantages of high reaction stereoselectivity, high product yield, simple halogen source, easy operation process control, low production cost, green and environmentally friendly reaction, and high catalytic activity.

[0073] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of halogenases in the catalytic synthesis of 2'-halonucleotides, characterized in that, The halogenase is any one of NSH1, NSH2, NSH3, NSH4, and NSH5, wherein the amino acid sequence of NSH1 is shown in SEQ ID NO.1, the amino acid sequence of NSH2 is shown in SEQ ID NO.2, the amino acid sequence of NSH3 is shown in SEQ ID NO.3, the amino acid sequence of NSH4 is shown in SEQ ID NO.4, and the amino acid sequence of NSH5 is shown in SEQ ID NO.

5. The substrate used in the catalysis is any one of 2'-deoxyguanosine, 2'-deoxyinosine, and 2'-deoxyadenosine; The catalytic reaction system comprises: any one of halogenases NSH1, NSH2, NSH3, NSH4 and NSH5, ferrous ions, L-ascorbic acid, α-ketoglutarate, halide salts and the substrates described above; The halide is a chloride salt.

2. The application of halogenases in the catalytic synthesis of 2'-halonucleotides, characterized in that, The halogenase is NSH1 or NSH2, the amino acid sequence of NSH1 is shown in SEQ ID NO.1, and the amino acid sequence of NSH2 is shown in SEQ ID NO.2; The substrate used in the catalysis is 2'-deoxyadenosine; The catalytic reaction system comprises: halogenase NSH1 or NSH2, ferrous ions, L-ascorbic acid, α-ketoglutaric acid, halide salt, and the substrate; The halide is a bromine salt.

3. The application of halogenases in the catalytic synthesis of 2'-halonucleotides, characterized in that, The halogenase is any one of NSH3, NSH4 and NSH5, the amino acid sequence of NSH3 is shown in SEQ ID NO.3, the amino acid sequence of NSH4 is shown in SEQ ID NO.4, and the amino acid sequence of NSH5 is shown in SEQ ID NO.5; The substrate used in the catalysis is 2'-deoxyguanosine; The catalytic reaction system comprises: any one of halogenases NSH3, NSH4 and NSH5, ferrous ions, L-ascorbic acid, α-ketoglutaric acid, halide salts and the substrates described above; The halide is a bromine salt.

4. The application according to claim 1, characterized in that, The expressed and purified halogenases NSH1, NSH2, NSH3, NSH4, or NSH5 are used as biocatalysts to catalyze the halogenation of nucleoside substrates.

5. The application according to claim 1, characterized in that, The chloride salt is sodium chloride or potassium chloride.

6. The application according to claim 2 or 3, characterized in that, The bromide salt is sodium bromide or potassium bromide.

7. The application according to claim 4, characterized in that, The reaction solution is a buffer solution with a pH of 6-8, and the reaction temperature is 20-40℃.

8. The application according to claim 4, characterized in that, The expression steps include: constructing the gene sequence of the halogenase into an expression vector to obtain a recombinant vector, and transforming it into Escherichia coli to induce the expression of the halogenase protein.

9. The application according to claim 4, characterized in that, The purification process involved purifying the halogenated enzyme protein using a nickel column.

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