Biocatalytic synthesis method of fluoro nucleoside compound

Abstract

The invention relates to a biological catalytic synthesis method of a fluoro nucleoside compound, and belongs to the technical field of genetic engineering and biological catalysis. In the high-efficiency and low-cost production process of the fluoro-nucleoside provided by the invention, the enzyme catalyst containing thymine nucleoside phosphorylase and purine nucleoside phosphorylase is subjected to heating pretreatment, so that when the enzyme catalyst is used as the catalyst in the preparation process of the fluoro-nucleoside, a protein purification step with complex procedures and high cost is not needed; the method provided by the invention can be directly used for production and preparation of fluoro-nucleoside, has a very good catalytic effect, can obviously reduce the cost of biocatalytic synthesis of fluoro-nucleoside, also has certain reference significance for treatment of enzyme preparations in other biocatalytic synthesis, and has wide production and application values.

Description

Technical Field

[0001] The present invention relates to a method for biocatalytic synthesis of fluorinated nucleoside compounds, belonging to the technical fields of genetic engineering and biocatalysis. Background Art

[0002] Monomers and their complexes of nucleoside analogs have been widely used in the treatment of cancer and antiviral. In order to design effective, selective and non-toxic drugs, various strategies have been designed in the prior art, involving the modification of natural nucleosides, especially the change of the carbohydrate moiety. For example, the modification of the carbon atom at the 2-position of the pentafuranose ring is a common structure of artificial nucleoside analogs and has great pharmaceutical potential (2021, Process Biochemistry), and according to research, C2'-fluorinated nucleosides are more effective as components of antisense oligonucleotides and small interfering RNAs (see Patent CN111534493B).

[0003] However, the high cost of nucleoside analog drugs hinders their extensive biological research and therapeutic applications. The traditional chemical synthesis method not only has many steps, takes a long time, produces environmentally polluting reagents, but also requires glycosyl activation with high technical difficulty, control of the stereoselectivity and regioselectivity of glycoside formation, and protection and deprotection of sensitive functional groups (see CN115667280A, CN101830955B).

[0004] Therefore, using biocatalysis to effectively synthesize nucleoside analogs under environmentally friendly conditions has become a more attractive alternative approach, which makes the synthesis process more effective and green and clean (see CN115725676). By using biocatalysis to synthesize nucleoside analogs, the mutual conversion between purine nucleosides and pyrimidine nucleosides can be achieved through the co-catalysis of thymidine phosphorylase (TPase; EC2.4.2.4) and purine nucleoside phosphorylase (PNPase; EC 2.4.2.1), so as to synthesize nucleoside compounds such as adenosine, inosine, uridine, guanosine and their analogs (see CN113373100A).

[0005] At present, methods for the biocatalytic synthesis of nucleoside analogs have received extensive attention and research reports. However, the existing technology for this synthesis pathway still faces the following two main problems: First, the high cost of using pure enzymes or immobilized enzymes, as well as the high technical difficulty of enzyme modification or construction of co-expression vectors. Second, there are problems of low production efficiency, such as low conversion efficiency of the reaction system and low substrate concentration. Therefore, the present invention provides a high-efficiency production process for nucleoside analogs to solve the problems of high production cost and low production efficiency in the existing technology. Summary of the Invention

[0006] To solve the deficiencies in the existing technology, the present invention provides a method for the biocatalytic synthesis of fluorinated nucleoside compounds. In this production process, the enzyme catalyst containing thymidine phosphorylase and purine nucleoside phosphorylase is subjected to heat pretreatment. When used as a catalyst in the preparation of fluorinated nucleosides, it does not require a protein purification step with a complex procedure and high cost, and can be directly used for the production and preparation of fluorinated nucleosides. It has a very good catalytic effect and can significantly reduce the process cost of biocatalytic synthesis of fluorinated nucleosides.

[0007] According to one aspect of the present invention, there is provided a method for the biocatalytic synthesis of fluorinated nucleoside compounds, the method comprising the following steps:

[0008] 1) Ferment and culture an engineered bacterium expressing thymidine phosphorylase and purine nucleoside phosphorylase to obtain the bacterial liquid after the completion of the culture;

[0009] 2) Prepare an enzyme catalyst using the bacterial liquid obtained in step 1);

[0010] 3) Subject the enzyme catalyst obtained in step 2) to heat pretreatment;

[0011] 4) Use the enzyme catalyst obtained in step 3) to catalyze the synthesis of fluorinated nucleosides.

[0012] The engineered bacterium can be an engineered bacterium commonly used in the art, especially an engineered bacterium for expressing thymidine phosphorylase and purine nucleoside phosphorylase. For example, it can specifically be one or more of Escherichia coli, yeast, Bacillus subtilis, etc. Those skilled in the art can make a choice according to needs and actual situations.

[0013] The fluorinated nucleosides can be selected from a variety of fluorinated adenosine, fluorinated guanosine, fluorinated thymidine, fluorinated cytidine, and their analogs, etc. It can be understood by those skilled in the art that the process capable of obtaining fluorinated nucleosides or their analogs under the catalytic action of thymidine phosphorylase and purine nucleoside phosphorylase should be included within the scope of this solution.

[0014] The present invention does not specifically limit the sources of the thymidine phosphorylase and purine nucleoside phosphorylase, and those skilled in the art can make selections according to needs. For example, in some embodiments of the present invention, the enzymes used are TPase and PNPase from Escherichia coli. It is certain that in addition, TPase and PNPase from the following sources can also catalyze the reaction. For example, CsaTPase (from Cronobacter sakazakii, A7MGA9.1), VchTPase (from Vibrio cholerae, Q9KPL8.2), CviTPase (from Chromobacterium violaceum, Q7NRT0.1) and YenPNPase (from Yersinia enterocolitica, A1JJA0.1), PprPNPase (from Photobacterium profundum, Q6LUH1.1), CnoPNPase (from Clostridium novyi, A0Q1A0.1).

[0015] Specifically, in the embodiments of the present invention, thymidine phosphorylase TPase (CsaTPase, VchTPase, CviTPase) and purine nucleoside phosphorylase PNPase (YenPNPase, PprPNPase, CnoPNPase) are also used for pairwise combination reactions, namely CsaTPase&YenPNPase, CsaTPase&PprPNPase, CsaTPase&CnoPNPase, VchTPase&YenPNPase, VchTPase&PprPNPase, VchTPase&CnoPNPase, CviTPase&YenPNPase, CviTPase&PprPNPase, CviTPase&CnoPNPase.

[0016] Optionally, the conditions for the heat pretreatment are: the temperature is not lower than 40°C and the treatment duration is not lower than 30 min.

[0017] Optionally, the temperature is selected from 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C and any value therebetween, and the treatment duration is selected from 30 min, 35 min, 40 min, 45 min, 50 min, 1 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2.0 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h, 2.5 h, 2.6 h, 2.7 h, 2.8 h, 2.9 h, 3.0 h, 3.1 h, 3.2 h, 3.3 h, 3.4 h, 3.5 h, 3.6 h, 3.7 h, 3.8 h, 3.9 h, 4.0 h, 4.1 h, 4.2 h, 4.3 h, 4.4 h, 4.5 h, 4.6 h, 4.7 h, 4.8 h, 4.9 h, 5.0 h, 5.1 h, 5.2 h, 5.3 h, 5.4 h, 5.5 h, 5.6 h, 5.7 h, 5.8 h, 5.9 h, 6.0 h and any value therebetween.

[0018] Preferably, the conditions for the heat pretreatment are: the temperature is not lower than 50°C and the treatment duration is not lower than 1 h.

[0019] Optionally, when the thymidine phosphorylase and / or purine nucleoside phosphorylase is from Escherichia coli, the temperature in the conditions for the heat pretreatment is not higher than 60°C.

[0020] For thymidine phosphorylase and / or purine nucleoside phosphorylase from Escherichia coli, when the temperature is higher than 60°C, its activity will be affected. Therefore, in the conditions for the heat pretreatment, the temperature only needs to maintain the enzyme activity of thymidine phosphorylase and purine nucleoside phosphorylase. For example, for thymidine phosphorylase and / or purine nucleoside phosphorylase from some thermophilic bacteria, the optimal temperature of the enzyme is higher than that of enzymes from other bacteria. Those skilled in the art can understand that in the conditions for the heat pretreatment in the present invention, the temperature only needs to satisfy ensuring the enzyme activity of thymidine phosphorylase and / or purine nucleoside phosphorylase, and those skilled in the art can obtain this through testing according to the data of the prior art or through conventional single-factor experiments.

[0021] For example, the optimal temperature of HeTPase from Halomonas elongata is close to 70°C. Therefore, the temperature for heat pretreatment can be higher than 60°C. A person skilled in the art can obtain its appropriate or other available heat pretreatment temperatures through routine experiments. Another example is GtPNPase from Geobacillus thermoglucosidasius, whose optimal temperature is close to 60°C. Therefore, the conditions for its heat pretreatment can also be obtained by a person skilled in the art based on common knowledge of the enzyme's working temperature and combined with routine experiments.

[0022] Optionally, in step 4), based on a substrate concentration of 40 mM as the standard, when the enzyme catalyst is a crude enzyme solution catalyst, the concentration of thymidine phosphorylase is not less than 1 g / L, and the concentration of purine nucleoside phosphorylase is not less than 3 g / L.

[0023] It should be noted that the above substrate concentration refers to the reaction substrate used in the fluorinated nucleoside production process, and a person skilled in the art can understand its specific meaning. Taking the production of fluorinated nucleosides with a reaction principle as an example, here the substrate can refer to the concentration of only one of the substrates, such as 2'-fluoro-2'-deoxyuridine, or the concentration of only one of the substrates, such as adenine, or the sum of the concentrations of the substrates required for the fluorinated nucleoside reaction. Figure 1 Taking the production of fluorinated nucleosides with a reaction principle as an example, here the substrate can refer to the concentration of only one of the substrates, such as 2'-fluoro-2'-deoxyuridine, or the concentration of only one of the substrates, such as adenine, or the sum of the concentrations of the substrates required for the fluorinated nucleoside reaction.

[0024] Optionally, in step 4), the concentration ratio of the purine nucleoside phosphorylase to the thymidine phosphorylase is not less than 2.

[0025] Optionally, in step 4), the concentration ratio of the thymidine phosphorylase to the purine nucleoside phosphorylase is 1:W, where W is selected from 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 7.0, 8.0, 9.0, 10.0 and any value therebetween.

[0026] Optionally, in step 4), when the enzyme catalyst is a crude enzyme solution catalyst, the concentration ratio of the purine nucleoside phosphorylase to the thymidine phosphorylase is not less than 2.

[0027] Optionally, in step 4), when the concentration of the thymidine phosphorylase is 1 - 32 g / L, the concentration of the purine nucleoside phosphorylase is 3 - 160 g / L.

[0028] Optionally, the concentration of the thymidine phosphorylase is selected from 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, and any value therebetween.

[0029] Optionally, the concentration of the purine nucleoside phosphorylase is selected from 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, 100 g / L, 110 g / L, 120 g / L, 130 g / L, 140 g / L, 150 g / L, 160 g / L, and any value therebetween.

[0030] Optionally, in step 4), the concentration of the substrate is not less than 40 mM.

[0031] Optionally, in step 4), the concentration of the substrate is not less than 80 mM.

[0032] Optionally, in step 4), the concentration of the substrate is not less than 160 mM.

[0033] Optionally, in step 4), the concentration of the substrate is not less than 240 mM.

[0034] Optionally, in step 4), the concentration of the substrate is not less than 320 mM. When in accordance with Figure 1When the reaction is carried out for production, when the substrate concentration is calculated based on 2'-fluoro-2'-deoxyuridine, this scheme can expand the substrate concentration to more than 320 mM, improve the production volume of a single batch production, and has important significance for industrial production applications.

[0035] Optionally, in step 4), the concentration of the substrate is not less than 480 mM. It can be seen that when the enzyme catalyst is in the form of a whole cell catalyst in this scheme, the substrate concentration can be further expanded to 480 mM.

[0036] Optionally, in step 4), the concentration of the substrate is not less than 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 310 mM, 320 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 380 mM, 390 mM, 400 mM, 410 mM, 420 mM, 430 mM, 440 mM, 450 mM, 460 mM, 470 mM, 480 mM, 490 mM, 500 mM and any value therebetween.

[0037] Optionally, based on a substrate concentration of 40 mM, when the enzyme catalyst is a whole cell catalyst, the concentration of the whole cell catalyst expressing purine nucleoside phosphorylase is not less than 5 OD, and the concentration of the whole cell catalyst expressing thymidine phosphorylase is not less than 15 OD.

[0038] It should be noted that the purine nucleoside phosphorylase and the thymidine phosphorylase can be respectively constructed into two strains for expression. As mentioned above, the two strains can use the same strain, such as Escherichia coli, or other strains, or a combination of different strains, such as common engineering bacteria like yeast and Bacillus, or a strain that simultaneously expresses purine nucleoside phosphorylase and thymidine phosphorylase can also be constructed, and two whole cell catalysts that respectively express purine nucleoside phosphorylase and thymidine phosphorylase are added simultaneously during synthesis production.

[0039] According to another aspect of the present invention, a preparation method of an enzyme catalyst is provided. The preparation method includes the following steps:

[0040] 1) Ferment and culture an engineering bacterium expressing thymidine phosphorylase and purine nucleoside phosphorylase to obtain a bacterial liquid after the completion of cultivation;

[0041] 2) Use the bacterial liquid obtained in step 1) to prepare a to-be-treated enzyme catalyst, and the to-be-treated enzyme catalyst is one or both of a whole cell catalyst and a crude enzyme liquid catalyst;

[0042] 3) Perform heat pretreatment on the to-be-treated enzyme catalyst obtained in step 2) to obtain the enzyme catalyst;

[0043] The conditions for the heating pretreatment are as follows: the temperature is not lower than 40°C, and the treatment duration is not lower than 30 min.

[0044] Optionally, the temperature is selected from 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C and any value therebetween, and the treatment duration is selected from 30 min, 35 min, 40 min, 45 min, 50 min, 1 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2.0 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h, 2.5 h, 2.6 h, 2.7 h, 2.8 h, 2.9 h, 3.0 h, 3.1 h, 3.2 h, 3.3 h, 3.4 h, 3.5 h, 3.6 h, 3.7 h, 3.8 h, 3.9 h, 4.0 h, 4.1 h, 4.2 h, 4.3 h, 4.4 h, 4.5 h, 4.6 h, 4.7 h, 4.8 h, 4.9 h, 5.0 h, 5.1 h, 5.2 h, 5.3 h, 5.4 h, 5.5 h, 5.6 h, 5.7 h, 5.8 h, 5.9 h, 6.0 h and any value therebetween.

[0045] Preferably, the conditions for the heating pretreatment are as follows: the temperature is not lower than 50°C, and the treatment duration is not lower than 1 h.

[0046] Optionally, when the thymidine phosphorylase and / or purine nucleoside phosphorylase is of Escherichia coli origin, the temperature in the conditions for the heating pretreatment is not higher than 60°C.

[0047] According to another aspect of the present invention, there is provided an enzyme catalyst obtained by the preparation method of the above enzyme catalyst.

[0048] According to the last aspect of the present invention, there is provided the use of the above enzyme catalyst in the production of fluorinated nucleosides.

[0049] The beneficial effects of the present invention include but are not limited to:

[0050] 1. The biocatalytic synthesis method of fluorinated nucleoside compounds according to the present invention can maintain the product formation rate above 60% while greatly reducing the production cost, which is very close to the scheme using high-cost pure enzymes. Moreover, the substrate concentration can be increased to more than 320 mM, improving the substrate concentration in a single batch reaction, thereby greatly enhancing the production efficiency of the product in each batch.

[0051] 2. For the biocatalytic synthesis method of fluorinated nucleoside compounds according to the present invention, in order to further reduce the production cost, experiments were conducted on the lowest possible enzyme-substrate ratio on the basis of ensuring that the product formation rate is above 60%. When TPase is not less than 1 g / L and PNPase is not less than 3 g / L, not only can the highest possible product formation rate be obtained, but also the dosage of enzyme preparations can be reduced, thereby reducing the production cost.

[0052] 3. For the biocatalytic synthesis method of fluorinated nucleoside compounds according to the present invention, by adding a heat treatment step during the preparation of enzyme preparations, the product formation rate close to that of using pure enzymes can be achieved. This not only reduces the production cost, but also simplifies the production process, and can achieve very excellent results. It is suitable for the industrial production of fluorinated nucleosides and has broad application scenarios and economic value.

[0053] 4. The biocatalytic synthesis method of fluorinated nucleoside compounds according to the present invention is different from the technical idea of repeatedly purifying the enzyme catalyst for fluorinated nucleoside synthesis in the prior art. Instead, it improves from other factors in the preparation of enzyme preparations, and has achieved a technical progress close to the catalytic effect of pure enzymes, and can also greatly reduce the production cost. This also has reference significance for the preparation of enzyme preparations in other biocatalytic syntheses. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The drawings described herein are used to provide a further understanding of the present invention and form a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0055] Figure 1 It is a schematic diagram of the enzymatic reaction for generating fluoroadenosine involved in the specific embodiment of the present invention;

[0056] Figure 2 It is a schematic diagram of the comparison results of the product formation rates of crude enzymes 1, 2, 3 groups and the pure enzyme group involved in Comparative Example 1 of the present invention;

[0057] Figure 3 It is a schematic diagram of the comparison results of the detection peak diagrams of the crude enzyme and the pure enzyme involved in Comparative Example 1 of the present invention;

[0058] Figure 4Schematic diagram of the comparison result of the product formation rate between the whole-cell catalyst and the pure enzyme involved in Comparative Example 2 of the present invention;

[0059] Figure 5 Schematic diagram of the comparison result of the product formation rate under different heating pretreatment conditions involved in Example 2 of the present invention;

[0060] Figure 6 Schematic diagram of the enzymatic reaction to produce fluoroguanosine involved in Example 7 of the present invention;

[0061] Figure 7 Schematic diagram of the enzymatic reaction to produce fluorothymidine involved in Example 7 of the present invention. Detailed Description of the Invention

[0062] The present invention will be described in detail below with reference to the embodiments. However, the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials in the embodiments of the present invention are purchased through commercial channels.

[0063] As Figure 1 shown, the embodiments of the present invention are centered around the biotechnology of enzymatic synthesis of fluoronucleosides (hereinafter referred to as 2’F-dA). Specifically, TPase and PNPase are used as biocatalysts to catalyze the transglycosylation reaction of substrate S1 (2’-fluoro-2’-deoxyuridine, hereinafter referred to as 2’F-dU) and substrate S3 (adenine) to produce the target product P3 (2’-fluoro-2’-deoxyadenosine, hereinafter referred to as 2’F-dA) and the accompanying product P1 (uracil), where phosphate is the substrate in the first step and the product in the second step of the double-enzyme catalytic reaction.

[0064] The present invention will be described below with specific examples and experimental examples. It should be noted that the meanings of the product formation rate, the yield of 2’F-dA, or the yield are the same in the following text, and those skilled in the art can understand them conventionally.

[0065] Example 1 Preparation of Enzyme Catalyst

[0066] 1) Construction of expression host: In this protocol, the nucleotide sequence of the coding gene of TPase used is shown in SEQ ID No.1, and the nucleotide sequence of the coding gene of PNPase is shown in SEQ ID No.2. The two sequences are respectively ligated to the vector pET-28b. As is known to those skilled in the art, the nucleotide sequences of the coding genes of TPase and PNPase in this example are not limited to SEQ ID No.1 or SEQ ID No.2, and any nucleotide sequence capable of encoding TPase or PNPase can be used here. Then the expression vectors of TPase and PNPase are respectively introduced into host cells. Finally, the transformants are spread on an LB plate medium containing kanamycin for culture and screening. After sequencing and identification, an expression host is obtained.

[0067] 2) Cultivation of expression host: The host cells of TPase and PNPase are respectively inoculated into an LB sterile culture solution containing 50 μg / ml kanamycin and cultured in a shaker at 37°C and 220 rpm for more than 12 h. 4 ml of the activated bacterial liquid is respectively taken and inoculated into a 2000 ml conical flask containing 400 ml of LB medium containing 50 μg / ml kanamycin. Cultivate in a shaker at 37°C and 220 rpm until the OD value reaches 0.6 - 0.8. In a laminar flow hood, IPTG is added to a final concentration of 0.15 mM, and induced culture is carried out in a shaker at 20°C and 180 rpm for 18 h.

[0068] 3) Preparation of enzyme catalyst: a) Whole-cell enzyme catalyst: The cultured bacterial liquid is centrifuged for 15 min, the supernatant is removed, and it is stored in a -20°C refrigerator for later use; b) Crude enzyme liquid enzyme catalyst: Set the power of the ultrasonic crusher to 1000 W, ultrasonic for 3 s, with an interval of 2 s, and a total time of 20 min. The crushed bacterial liquid is centrifuged for 20 min, and the supernatant is taken to measure the total protein concentration for later use; c) Purified enzyme: On the basis of the preparation of the crude enzyme liquid, the supernatant is incubated with a nickel column overnight, washed with 50 mM imidazole to remove impurities, and eluted with 300 mM imidazole. The eluate is concentrated with a 10 kDa ultrafiltration tube and imidazole is removed for later use.

[0069] Comparative Example 1 The crude enzyme liquid enzyme catalyst is used for the biosynthesis of 2’F-dA

[0070] The reaction system was set to 0.5 ml, the reaction buffer was potassium phosphate buffer at 100 mM and pH 7.0, the concentration of substrate 2’F-dU was 40 mM, and the concentration of substrate adenine was 44 mM. Then the crude TPase enzyme solution was added, and three concentration gradients were set. The concentrations of groups 1, 2, and 3 were 0.8, 1.6, and 2.5 g / L respectively. After reacting at 50 °C and 240 rpm for 4 h, the crude PNPase enzyme solution was added, and the corresponding concentration gradients of groups 1, 2, and 3 were 5, 10, and 15 g / L. At the same time, the reaction catalyzed by the pure enzyme solution was used as a positive control, with the concentration of pure enzyme solution TPase being 0.8 g / L and the concentration of PNPase being 5 g / L. After reacting for 24 h, samples were taken respectively, and the consumption of the substrate and the generation of the product were detected by Agilent 1260 Infinity II HPLC using an ultraviolet detector at 254 nm. The liquid phase column used was Agilent C18 (250 mm x 4.6 mm x 5 μm), the flow rate was 1.0 ml / min, and the column temperature was 30 °C. Mobile phase A was pure acetonitrile, and mobile phase B was 0.1% phosphoric acid aqueous solution. It was run from 5% A and 95% B to 95% A and 5% B for 15 min.

[0071] The detection results are as Figure 2 shown. Compared with the product generation rate of 70.01% after 24 h in the pure enzyme group, the generation rates of the target product 2’F-dA in the crude enzyme groups 1, 2, and 3 were 42.89%, 40.88%, and 29.77% respectively. That is, the generation rate of the target product 2’F-dA was much lower than that in the pure enzyme group, and it decreased instead as the enzyme concentration increased. By analyzing the detection peak charts of the pure enzyme and the crude enzyme, as Figure 3 shown, it was shown that there was an additional impurity peak (4.619 min) in the reaction solution of the crude enzyme. Based on the above, it was speculated that there was a substance in the crude enzyme solution that could degrade the target product 2’F-dA.

[0072] Comparative Example 2 Whole cells were used for the biosynthesis of 2’F-dA

[0073] The reaction system and component concentrations were set under the same conditions as in Comparative Example 1. The TPase whole cells used were 20 OD, and the PNPase cells were 100 OD. The reaction system detection was the same as in Comparative Example 1.

[0074] The detection results showed that compared with the pure enzyme, the product generation rate of the whole cells was only 46.07% after 24 h, and there was also an impurity peak at the same position (4.619 min) in the whole cell reaction solution. Similarly, it was speculated that there was a substance in the whole cells that could degrade the target product 2’F-dA.

[0075] Experimental Example 1 Heat pretreatment of host cells to inhibit the degradation of 2’F-dA

[0076] 1) Treat the host cells at different heating temperatures

[0077] The cell lysate of the host cells transformed with the empty vector pET-28b was pretreated for 1 h at five temperature gradients (40 °C, 50 °C, 60 °C, 70 °C, 80 °C) respectively, and then the pretreated cell lysate of the host cells was incubated for 1 h and 3 h respectively in the same reaction system (total volume 0.5 ml, reaction buffer was 100 mM potassium phosphate buffer at pH 7.0, substrate 2’F-dA 10 mM, protein concentration of the cell lysate was 20 g / L), and then detected by HPLC. At the same time, the cell lysate without heat treatment (pET-NoT) was used as the positive control group, and the group without adding crude enzyme (Buffer) was used as the negative control group.

[0078] Table 1

[0079]

[0080]

[0081] The detection results are shown in Table 1 above. In the untreated positive control group, 2’F-dA was largely converted into a miscellaneous peak at 4.619 min after 1 h and 3 h of reaction. The peak area of the miscellaneous peak in the sample (pET-40-1h) pretreated at 40 °C for 1 h and then incubated for 1 h decreased, and the peak area of the miscellaneous peak converted in the samples pretreated at 50 °C for 1 h and then incubated for 1 h or 3 h (pET-50-1h and pET-50-3h) was very small. No miscellaneous peaks were detected in the groups treated at 60 °C for 1 h (pET-60) and higher temperatures. In summary, treatment at 60 °C for 1 h and treatment at higher temperatures for 1 h can completely inhibit the generation of miscellaneous peaks, that is, inhibit the degradation of the target product 2’F-dA by host cells.

[0082] 2) Treatment of host cells with different heating times

[0083] The cell lysate of the host cells was heated at 50 °C for different times (1 h, 2 h, 3 h) respectively, and the rest of the reaction system was consistent with the experimental design of treating host cells with different heating temperatures in 1). Samples were taken for HPLC detection after incubation for 1 h, 3 h and 24 h respectively.

[0084] Table 2

[0085] Sample Group\Peak Area 2’F-dA Impurity Peak 50-3-1h 7471.453 0 50-2-1h 7494.603 0 50-1-1h 8255.66 27.068 50-0-1h 131.74 5491.57 Buffer-1h 8421.749 0 50-3-3h 9836.687 0 50-2-3h 7433.07 15.452 50-1-3h 7110.609 40.408 50-0-3h 60.2 5713.675 Buffer-3h 7855.73 0 50-3-24h 7589.689 0 50-2-24h 7376.83 17.786 50-1-24h 7251.957 33.947 50-0-24h 23.896 5612.769 Buffer-24h 7665.631 0

[0086] The detection results are shown in Table 2. Compared with the untreated control group (50-0), samples incubated for 1 h, 3 h, and 24 h after treatment at 50 °C for 1 h (50-1-1 h, 50-1-3 h, 50-1-24 h) all produced miscellaneous peaks. The sample treated at 50 °C for 2 h showed miscellaneous peaks only after incubation for 3 h (50-2-3 h), and the sample treated at 50 °C for 3 h still did not show miscellaneous peaks after incubation for 24 h, indicating that treatment at 50 °C for 3 h can completely inhibit the generation of miscellaneous peaks.

[0087] Example 2: Heating pretreatment of the catalyst can effectively improve the reaction yield

[0088] Using the two heating pretreatment conditions screened in Experimental Example 1 that can inhibit the generation of miscellaneous peaks, namely 50 °C - 3 h and 60 °C - 1 h, the crude enzyme solutions of TPase and PNPase were pretreated respectively. The reaction system was set to 1 ml, the reaction buffer was potassium phosphate buffer at 100 mM and pH 7.0, the concentration of substrate 2’F-dU was 10 mM, the concentration of substrate adenine was 11 mM, the concentration of the crude enzyme solution of TPase was 1 g / L, and the concentration of the crude enzyme solution of PNPase was 5 g / L. Then the heated pretreated crude enzyme solution of TPase was added, and after reacting at 50 °C and 240 rpm for 4 h, the crude enzyme solution of PNPase was added, and then samples were taken after reacting for 72 h for HPLC detection.

[0089] The results showed that compared with the 2’F-dA production rate of 44.92% in the non-preheated treatment group, the production rate of the 50 °C - 3 h group was as high as 64.23%, effectively improving the reaction yield, while almost no production rate was detected in the 60 °C - 1 h group.

[0090] Example 3: Optimization of the reaction system of preheated crude enzyme solution

[0091] The crude enzyme solutions of TPase and PNPase pretreated at 50 °C for 3 h were used for the following reactions.

[0092] 1) Determination of the ratio of enzyme to substrate concentration in the reaction system

[0093] The reaction system was set to 1 ml, the reaction buffer was potassium phosphate buffer at 100 mM and pH 7.0, the concentration of substrate 2’F-dU was 40 mM, and the concentration of substrate adenine was 44 mM. Then the crude enzyme solution of TPase was added, and four concentration gradients were set. The concentrations of the 1st - 4th groups were 4, 2, 1, and 0.5 g / L respectively. After reacting at 50 °C and 240 rpm for 4 h, the crude enzyme solution of PNPase was added, and the corresponding concentration gradients of PNPase in the 1st - 4th groups were 20, 10, 5, and 2.5 g / L. Samples were taken respectively after reacting for 72 h and detected by HPLC.

[0094] The detection results are as Figure 4, the yields of the target product 2’F-dA in Group 1, Group 2, and Group 3 were basically the same, generally around 60%. As the enzyme concentration decreased, the yield of Group 4 decreased sharply. Therefore, the enzyme concentration combination of TPase and PNPase in Group 3 (1 g / L and 5 g / L) could maintain a relatively high product formation rate in the existing system.

[0095] 2) Optimization of the ratio of the two enzymes in the reaction system

[0096] Set the reaction system to 1 ml, the reaction buffer to 100 mM potassium phosphate buffer at pH 7.0, the concentration of substrate 2’F-dU to 40 mM, and the concentration of substrate adenine to 44 mM. Then add the crude TPase enzyme solution with a concentration of 1 g / L to all of them. After reacting at 50 °C and 240 rpm for 4 h, add the crude PNPase enzyme solution with different concentration gradients. Six concentration gradients are set, and the concentrations of Group 1 - 6 are 1, 2, 3, 4, 5, and 6 g / L respectively. After reacting for 72 h, sample respectively and detect by HPLC.

[0097] Detection results Figure 5 The results showed that the yields of Group 3, Group 4, Group 5, and Group 6 were all higher than 60%, and decreased slowly with the decrease of the PNPase concentration; while for Group 2 and Group 1, the yields decreased sharply (<30%) with the decrease of the enzyme concentration. Therefore, the enzyme concentration combination of TPase and PNPase in Group 3 (1 g / L and 3 g / L) could maintain a relatively high yield in the existing system.

[0098] 3) Expansion of the substrate concentration in the reaction system

[0099] Set the reaction system to 1 ml, the reaction buffer to 100 mM potassium phosphate buffer at pH 7.0. On the premise of fixing the enzyme-substrate ratio, increase the substrate concentration in gradients. A total of six groups are set. In Group 1, the concentration of substrate 2’F-dU is 40 mM, the concentration of substrate adenine is 44 mM, the concentration of the crude TPase enzyme solution is 1 g / L, and the concentration of the crude PNPase enzyme solution is 3 g / L. The ratios of substrate 2’F-dU, adenine to enzymes TPase and PNPase in Group 2, 3, 4, and 5 are 80:88:2:6, 160:176:4:12, 240:264:6:18, and 320:352:8:24 respectively. After reacting for 96 h, sample respectively and detect by HPLC.

[0100] The detection results showed that after the substrate concentration was increased, the yields of each group were basically the same, which were 66.87%, 66.60%, 69.88%, 65.37%, and 63.54% respectively. Therefore, in the crude enzyme solution reaction system, on the premise of ensuring the yield, the concentration of substrate 2’F-dU can be increased to 320 mM.

[0101] Example 4 Synthesis of 2’F-dA Using Heat-Pretreated Whole-Cell Catalysts

[0102] The whole cells of TPase and PNPase were pretreated under heating conditions of 50°C for 3 h to prepare the catalysts. The reaction system was set to 1 ml, and the reaction buffer was potassium phosphate buffer at 100 mM and pH 7.0. The reaction was divided into 5 groups. In group 1, the concentration of substrate 2’F-dU was 160 mM, the concentration of substrate adenine was 176 mM, the whole-cell concentration of TPase was 24 OD, and the whole-cell concentration of PNPase was 72 OD. The ratios of substrate 2’F-dU, adenine to the whole cells of enzymes TPase and PNPase in groups 2, 3, 4, and 5 were 320:352:48:144, 480:528:72:216, 640:704:96:288, and 800:880:120:360, respectively. Samples were taken after 72 h of reaction and detected by HPLC.

[0103] The detection results showed that the yields of 2’F-dA in groups 1, 2, and 3 were basically the same, which were 60.12%, 62.54%, and 59.92%, respectively. The yields in groups 4 and 5 were lower, which were 41.36% and 36.75%, respectively. Therefore, in the whole-cell reaction system, the concentration of substrate 2’F-dU can be increased to 480 mM and still maintain a relatively high yield of 2’F-dA.

[0104] Example 5 Scale-Up Production of 2’F-dA by Whole-Cell Catalysis (500 ml)

[0105] The reaction system was set to 500 ml, the reaction buffer was potassium phosphate buffer at 100 mM and pH 7.0, the concentration of substrate 2’F-dU was 160 mM, the concentration of substrate adenine was 176 mM. The whole-cell catalyst of TPase was 24 OD and the whole-cell catalyst of PNPase was 72 OD as described in Example 4. Samples were taken after 72 h of reaction and detected by HPLC.

[0106] The detection results showed that the yield of 2’F-dA reached 64.0% after 72 h of reaction.

[0107] Example 6 Using TPase and PNPase from Other Sources

[0108] In the previous examples, the enzymes used were TPase and PNPase from Escherichia coli. In this example, pairwise combination reactions were carried out using TPase (CsaTPase, VchTPase, CviTPase) and PNPase (YenPNPase, PprPNPase, CnoPNPase), namely CsaTPase&YenPNPase, CsaTPase&PprPNPase, CsaTPase&CnoPNPase, VchTPase&YenPNPase, VchTPase&PprPNPase, VchTPase&CnoPNPase, CviTPase&YenPNPase, CviTPase&PprPNPase, CviTPase&CnoPNPase, which were set as groups 1 - 9 respectively. The whole cells of TPase and PNPase pretreated at 50°C for 3 h were used as the experimental groups, and the enzymes without heat pretreatment were used as the control. The reaction system was set to 1 ml, the reaction buffer was potassium phosphate buffer at 100 mM and pH 7.0, the concentration of substrate 2’F-dU was 10 mM, the concentration of substrate adenine was 11 mM, the concentration of TPase was 6 OD, and the concentration of PNPase was 18 OD. First, the whole cells of the said TPase were added, and after reacting at 50°C and 240 rpm for 4 h, the whole cells of the said PNPase were added. Then, samples were taken after reacting for 96 h respectively, and the yield of 2’F-dA was detected by HPLC.

[0109] The results are shown in Table 3. The yields of 2’F-dA in the experimental groups (50°C - 3 h) of groups 1 - 5 and 7 - 9 were significantly higher than those in the control group, and the control group was the untreated group, indicating that the heat pretreatment process can be widely applied to TPase and PNPase from different sources.

[0110] Table 3 Experimental results of TPase and PNPase from different sources

[0111] Grouping 50℃-3h Control Group 1 46.21％ 17.51％ Group 2 27.02％ 6.74％ Group 3 51.93％ 43.08％ Group 4 20.43％ 2.12％ Group 5 11.17％ 7.19％ Group 6 29.09％ 31.38％ Group 7 36.24％ 1.18％ Group 8 20.78％ 1.60％ Group 9 40.39％ 37.05％

[0112] Example 7 Synthesis of other fluorinated nucleosides

[0113] TPase and PNPase can not only be used for Figure 1 the biosynthesis of 2’F-dA in Figure 6 but also for the synthesis of other fluorinated nucleosides, such as Figure 7 the synthesis of fluoroguanosine in

[0114] and the synthesis of fluorothymidine in Figure 6As shown below. The reaction system was set to 1 ml, the reaction buffer was potassium phosphate buffer at 100 mM and pH 7.0, the concentration of substrate 2’F-dU was 10 mM, the concentration of substrate guanine was 11 mM, the pure enzyme concentration of EcoTPase was 1 g / L, and the pure enzyme concentration of EcoPNPase was 5 g / L. EcoTPase was added first for the reaction. After reacting for 4 h at 50 °C and 240 rpm, EcoPNPase was added. After reacting for 72 h, samples were taken, and the yield of fluoroguanosine detected by HPLC was 3.21%.

[0115] The synthesis of fluorothymidine was catalyzed by EcoTPase, and the reaction process was as Figure 7 shown below. The reaction system was set to 1 ml, the reaction buffer was potassium phosphate buffer at 100 mM and pH 7.0, the concentration of substrate 2’F-dU was 1 mM, the concentration of substrate thymine was 10 mM, and the enzyme concentration of EcoTPase was 1 g / L. Samples were taken after reacting for 48 h at 50 °C and 240 rpm, and the yield of fluorothymidine detected by HPLC was 45.20%.

[0116] In the solution of the present invention, the inventors aimed at the problem in the enzymatic synthesis production of fluoronucleosides in the prior art that side reactions occurred and the target products were degraded when using crude enzymes or whole cells for catalysis, resulting in a very low production efficiency of the target products. Therefore, the synthesis method of the present invention's solution was provided, which could reduce production costs and simplify the production process, effectively avoid the formation of by-products, and improve the batch production efficiency.

[0117] The enzymatic synthesis technology for the efficient production of fluoronucleosides using crude enzymes or whole cells provided by the solution of the present invention also provided ideas for the production of other nucleoside analogs in related fields. It not only made obvious technological progress in the efficient catalytic synthesis using crude enzymes or whole cells, but also had guiding significance for the synthesis of other nucleoside analogs in the prior art.

[0118] As mentioned above, the above are only embodiments of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the technical idea and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for the biocatalytic synthesis of fluorinated nucleoside compounds, characterized in that, The method includes the following steps: 1) Ferment and culture the engineered bacteria expressing thymidine phosphorylase and purine nucleoside phosphorylase to obtain the bacterial liquid after the completion of the culture; 2) Prepare an enzyme catalyst using the bacterial liquid obtained in step 1); 3) Perform heat pretreatment on the enzyme catalyst obtained in step 2); 4) Catalyze the synthesis of fluorinated nucleoside using the enzyme catalyst obtained in step 3).

2. The method for the biocatalytic synthesis of fluorinated nucleoside compounds according to claim 1, characterized in that, The conditions for the heat pretreatment are: the temperature is not lower than 40 °C, and the treatment duration is not less than 30 min.

3. The method for the biocatalytic synthesis of fluorinated nucleoside compounds according to claim 2, characterized in that, When the thymidine phosphorylase and / or purine nucleoside phosphorylase is from Escherichia coli, the temperature in the conditions for the heat pretreatment is not higher than 60 °C.

4. The method for the biocatalytic synthesis of fluorinated nucleoside compounds according to claim 1, characterized in that, In step 4), based on the substrate concentration of 40 mM as the standard, when the enzyme catalyst is a crude enzyme liquid catalyst, the concentration of thymidine phosphorylase is not lower than 1 g / L, and the concentration of purine nucleoside phosphorylase is not lower than 3 g / L.

5. The method for the biocatalytic synthesis of fluorinated nucleoside compounds according to claim 4, characterized in that, In step 4), the concentration ratio of the purine nucleoside phosphorylase to the thymidine phosphorylase is not less than 2.

6. The method for the biocatalytic synthesis of fluorinated nucleoside compounds according to claim 5, characterized in that, In step 4), the concentration of the substrate is not lower than 40 mM.

7. The method for the biocatalytic synthesis of fluorinated nucleoside compounds according to claim 1, characterized in that, Based on the substrate concentration of 40 mM as the standard, when the enzyme catalyst is a whole-cell catalyst, the concentration of the whole-cell catalyst expressing the purine nucleoside phosphorylase is not lower than 5 OD, and the concentration of the whole-cell catalyst expressing the thymidine phosphorylase is not lower than 15 OD.

8. A method for preparing an enzyme catalyst, characterized in that, The preparation method includes the following steps: 1) Ferment and culture the engineered bacteria expressing thymidine phosphorylase and purine nucleoside phosphorylase to obtain the bacterial liquid after the completion of the culture; 2) Prepare the enzyme catalyst to be treated using the bacterial liquid obtained in step 1), and the enzyme catalyst to be treated is one or both of a whole-cell catalyst and a crude enzyme liquid catalyst; 3) Perform heat pretreatment on the enzyme catalyst to be treated obtained in step 2) to obtain the enzyme catalyst; The conditions for the heat pretreatment are: the temperature is 40 - 90 °C, and the treatment duration is not less than 30 min.

9. An enzyme catalyst obtained by the method for preparing an enzyme catalyst according to claim 8.

10. The application of the enzyme catalyst according to claim 9 in the production of fluorinated nucleosides.

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