阅读说明：本技术 一种酶法制备嘌呤霉素的方法 (Method for preparing puromycin by enzyme method ) 是由 赵弘 于铁妹 潘俊锋 刘建 于 2021-08-04 设计创作，主要内容包括：本发明涉及药物合成技术领域,特别涉及一种酶法制备嘌呤霉素的方法。该方法包括如下步骤：3-氨基-D-核糖在甲基硫核糖激酶与ATP作用下,生成β-氨基核糖-1-磷酸；β-氨基核糖-1-磷酸和N,N-二甲基嘌呤在嘌呤核苷磷酸化酶作用下,生成3-氨基-N,N-二甲基腺苷；3-氨基-N,N-二甲基腺苷和甲基酪氨酸在氨基酸连接酶与ATP作用下,生成嘌呤霉素。本发明针对嘌呤霉素发酵及化学制备方法局限,开发了其酶法制备工艺。相对于传统路线,该工艺具有路线短、转化率高、反应条件温和、环保等优点,这不仅显著的降低了嘌呤霉素生产成本,同时也提高了其工业生产中安全及绿色指数。(The invention relates to the technical field of drug synthesis, in particular to a method for preparing puromycin by an enzyme method. The method comprises the following steps: the 3-amino-D-ribose generates beta-aminoribose-1-phosphate under the action of methyl thioribose kinase and ATP; the 3-amino-N, N-dimethyl adenosine is generated by the beta-amino ribose-1-phosphate and the N, N-dimethyl purine under the action of purine nucleoside phosphorylase; the puromycin is generated by the action of 3-amino-N, N-dimethyl adenosine and methyl tyrosine under the action of amino acid ligase and ATP. Aiming at the limitation of fermentation and chemical preparation methods of puromycin, the invention develops an enzymatic preparation process of puromycin. Compared with the traditional route, the process has the advantages of short route, high conversion rate, mild reaction conditions, environmental protection and the like, so that the production cost of puromycin is remarkably reduced, and the safety and green index in industrial production are improved.)

1. A method for preparing puromycin by an enzymatic method is characterized by comprising the following steps:

the 3-amino-D-ribose generates beta-aminoribose-1-phosphate under the action of methyl thioribose kinase and ATP;

the 3-amino-N, N-dimethyl adenosine is generated by the beta-amino ribose-1-phosphate and the N, N-dimethyl purine under the action of purine nucleoside phosphorylase;

the puromycin is generated by the action of 3-amino-N, N-dimethyl adenosine and methyl tyrosine under the action of amino acid ligase and ATP.

2. The method of claim 1, wherein the purine nucleoside phosphorylase is PNP-a and/or PNP-b.

3. The method of claim 1, wherein the amino acid ligase is aalignase-1 and/or aalignase-2.

4. The method of claim 1, wherein the purine nucleoside phosphorylase is PNP-b and the amino acid ligase is aalignase-1.

5. The method according to claim 1, wherein the reaction system in which ATP is involved further comprises an ATP cycle system consisting of acetate kinase and acetyl phosphate.

6. The process according to any one of claims 1 to 5, characterized in that the process is a multicomponent one-pot process.

7. The method according to claim 6, wherein in the multi-component one-pot reaction system, the ratio of 3-amino-D-ribose, N-dimethylpurine, methyltyrosine, ATP, methylthioribokinase, purine nucleoside phosphorylase, and amino acid ligase is (50 to 150) mM: (50-150) mM: (50-150) mM: (1-2) mM: (1000-1500) U: (800-1500) U: (800-1500) U;

or 3-amino-D-ribose, N-dimethylpurine, methyltyrosine, ATP, acetyl phosphate, methyl thioribokinase, purine nucleoside phosphorylase, amino acid ligase and acetate kinase in a ratio of (50-150) mM: (50-150) mM: (50-150) mM: (1-2) mM: (100-300) mM: (1000-1500) U: (800-1500) U: (800-1500) U: (2000-4000) U.

8. The method of claim 6, wherein the multi-component one-pot reaction system further comprises an activator and a substrate co-solvent;

the activator is magnesium chloride, and the substrate cosolvent is isopropanol.

9. The method according to claim 8, wherein in the multi-component one-pot reaction system, the ratio of 3-amino-D-ribose, N-dimethylpurine, methyl tyrosine, ATP, an activator, a substrate cosolvent, methyl thioribokinase, purine nucleoside phosphorylase, and amino acid ligase is (50-150) mM: (50-150) mM: (50-150) mM: (1-2) mM: (5-15) mM: (100-150) mL: (1000-1500) U: (800-1500) U: (800-1500) U;

or the proportion of 3-amino-D-ribose, N-dimethylpurine, methyl tyrosine, ATP, acetyl phosphate, an activator, a substrate cosolvent, methyl thioribokinase, purine nucleoside phosphorylase, amino acid ligase and acetate kinase is (50-150) mM: (50-150) mM: (50-150) mM: (1-2) mM: (100-300) mM: (5-15) mM: (100-150) mL: (1000-1500) U: (800-1500) U: (800-1500) U: (2000-4000) U.

10. The method of claim 6, wherein the reaction conditions of the multicomponent one-pot process are: the pH value is 7.0-8.5, and the mixture is stirred for 2-10 hours at 15-30 ℃.

Technical Field

The invention relates to the technical field of drug synthesis, in particular to a method for preparing puromycin by an enzyme method.

Background

Puromycin (Puromycin) is an aminoglycoside antibiotic produced by fermentation and metabolism of Streptomyces albonubes (Streptomyces alboniger), and kills gram-positive bacteria, various animals, and insect cells by inhibiting protein synthesis. Effective in certain special cases; is commonly used to screen eukaryotic or prokaryotic polyclonal or monoclonal cells capable of expressing the pac gene (puror) by plasmid transfection/transformation, viral infection, and the like. Puromycin is not only used for screening of stable cell lines, but also for maintenance of stable cell lines. Puromycin is characterized by fast action on cells, and can kill 99 percent of cells which do not express pac genes within 2 days generally. In gram-positive bacterial, animal or insect cells, puromycin inhibits or kills the cells by inhibiting protein synthesis. The mechanism of action is that puromycin, an analogue of the 3' end of the aminoacyl-tRNA molecule, is able to bind to the a site of the ribosome and incorporate into the extended peptide chain. Puromycin, when bound to the A site, does not participate in any subsequent reaction, leading to premature termination of protein synthesis and release of the C-terminus.

The traditional preparation method of puromycin comprises a fermentation method and a chemical synthesis method:

1) a fermentation method: the streptomyces albus is used for fermentation production, but the yield is too low, and the purification process is complex and the production cost is high due to a plurality of byproducts.

2) Chemical synthesis method: norris j.robins reported the chemical synthesis of puromycin using 3-azido-adenosine as the starting material and 6-step chemical catalysis to produce puromycin with an overall yield of about 26%. The preparation of puromycin by a chemical synthesis method needs protection and deprotection of a plurality of functional groups, the whole route is long, and a plurality of toxic and harmful reagents (such as TMSCl, pyridine, DCC and the like) are needed in the chemical preparation process, so that the safety coefficient and the environmental compatibility in the production process are low, and the whole yield is not high, so that the final production cost is high.

With the public's attention to personal safety and natural environment protection in industrial production, the green chemical industry is a necessary trend in its development. As a part of biological catalysis, enzyme catalysis is compatible with the characteristics of high chemical catalysis concentration, environmental protection of fermentation production, mild conditions and the like, and is gradually becoming an important direction of green chemical development. Enzymatic preparation of puromycin is only reported.

Disclosure of Invention

In view of the above, the present invention provides a method for preparing puromycin by an enzymatic method. The method can realize the high-efficiency conversion of the aminoribose to the puromycin by utilizing three-step enzyme reaction, thereby having outstanding advantages in various aspects such as production cost, environmental protection, waste gas and waste water discharge and the like.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for preparing puromycin by an enzyme method, which comprises the following steps:

the 3-amino-D-ribose generates beta-aminoribose-1-phosphate under the action of methyl thioribose kinase and ATP;

the 3-amino-N, N-dimethyl adenosine is generated by the beta-amino ribose-1-phosphate and the N, N-dimethyl purine under the action of purine nucleoside phosphorylase;

the puromycin is generated by the action of 3-amino-N, N-dimethyl adenosine and methyl tyrosine under the action of amino acid ligase and ATP.

The invention utilizes the fact that methyl thioribokinase (Kinase, EC 2.7.1.100) can phosphorylate 3-amino-D-ribose to obtain corresponding beta-aminoribose-1-phosphate, then purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is converted into 3-amino-N, N-dimethyl adenosine under the action of N, N-dimethyl purine, and finally condensed with methylated tyrosine under the action of amino acid ligase (aaLigase, EC 6.3.2.28) to obtain the final puromycin.

In the present invention, the purine nucleoside phosphorylase is PNP-a and/or PNP-b.

In the present invention, the amino acid ligase is aalignase-1 and/or aalignase-2.

Preferably, the purine nucleoside phosphorylase is PNP-b and the amino acid ligase is aalignase-1.

Preferably, the reaction system involving ATP further includes an ATP cycle system composed of acetate kinase and acetyl phosphate.

In the first and third steps of reaction, Adenosine Triphosphate (ATP) is needed, so an ATP circulating system composed of acetate kinase (AK, EC 2.7.2.1)/acetyl phosphate (AcP) is added in the whole system, thereby greatly reducing the dosage of expensive adenosine triphosphate and further reducing the production cost.

Preferably, the preparation method is a multi-component one-pot method.

Preferably, in the multi-component one-pot reaction system, the proportion of 3-amino-D-ribose, N-dimethylpurine, methyl tyrosine, ATP, methyl thioribokinase, purine nucleoside phosphorylase and amino acid ligase is (50-150) mM: (50-150) mM: (50-150) mM: (1-2) mM: (1000-1500) U: (800-1500) U: (800-1500) U;

or 3-amino-D-ribose, N-dimethylpurine, methyltyrosine, ATP, acetyl phosphate, methyl thioribokinase, purine nucleoside phosphorylase, amino acid ligase and acetate kinase in a ratio of (50-150) mM: (50-150) mM: (50-150) mM: (1-2) mM: (100-300) mM: (1000-1500) U: (800-1500) U: (800-1500) U: (2000-4000) U.

Preferably, in the multi-component one-pot reaction system, the proportion of 3-amino-D-ribose, N-dimethylpurine, methyl tyrosine, ATP, methyl thioribokinase, purine nucleoside phosphorylase and amino acid ligase is (50-150) mM: (50-150) mM: (50-150) mM: 1.5 mM: (1000-1500) U: (800-1500) U: (800-1500) U;

or 3-amino-D-ribose, N-dimethylpurine, methyltyrosine, ATP, acetyl phosphate, methyl thioribokinase, purine nucleoside phosphorylase, amino acid ligase and acetate kinase in a ratio of (50-150) mM: (50-150) mM: (50-150) mM: 1.5 mM: (100-300) mM: (1000-1500) U: (800-1500) U: (800-1500) U: (2000-4000) U.

Preferably, the multi-component one-pot reaction system also comprises an activator and a substrate cosolvent;

preferably, the activator is magnesium chloride and the substrate co-solvent is isopropanol.

Preferably, in the multi-component one-pot reaction system, the proportion of 3-amino-D-ribose, N-dimethylpurine, methyl tyrosine, ATP, an activator, a substrate cosolvent, methyl thioribokinase, purine nucleoside phosphorylase and amino acid ligase is (50-150) mM: (50-150) mM: (50-150) mM: (1-2) mM: (5-15) mM: (100-150) mL: (1000-1500) U: (800-1500) U: (800-1500) U;

or the proportion of 3-amino-D-ribose, N-dimethylpurine, methyl tyrosine, ATP, acetyl phosphate, an activator, a substrate cosolvent, methyl thioribokinase, purine nucleoside phosphorylase, amino acid ligase and acetate kinase is (50-150) mM: (50-150) mM: (50-150) mM: (1-2) mM: (100-300) mM: (5-15) mM: (100-150) mL: (1000-1500) U: (800-1500) U: (800-1500) U: (2000-4000) U.

Preferably, in the multi-component one-pot reaction system, the proportion of 3-amino-D-ribose, N-dimethylpurine, methyl tyrosine, ATP, an activator, a substrate cosolvent, methyl thioribokinase, purine nucleoside phosphorylase and amino acid ligase is (50-150) mM: (50-150) mM: (50-150) mM: 1.5 mM: 10 mM: (100-150) mL: (1000-1500) U: (800-1500) U: (800-1500) U;

or the proportion of 3-amino-D-ribose, N-dimethylpurine, methyl tyrosine, ATP, acetyl phosphate, an activator, a substrate cosolvent, methyl thioribokinase, purine nucleoside phosphorylase, amino acid ligase and acetate kinase is (50-150) mM: (50-150) mM: (50-150) mM: 1.5 mM: (100-300) mM: 10 mM: (100-150) mL: (1000-1500) U: (800-1500) U: (800-1500) U: (2000-4000) U.

Preferably, the reaction conditions of the multicomponent one-pot process are: the pH value is 7.0-8.5, and the mixture is stirred for 2-10 hours at 15-30 ℃.

In the specific embodiment provided by the present invention, the reaction conditions of the multi-component one-pot process are: stirring for 4 hours at room temperature with the pH value of 7.0-8.5.

In the present invention, the reaction further comprises the steps of removing the enzyme, separating and purifying by using a nonpolar column, Seplite D101, and crystallizing.

Preferably, the crystallization is crystallization in an aqueous ethanol solution.

In the specific examples provided herein, the crystallization is in ethanol/water (3:2, v: v).

The invention provides a method for preparing puromycin by an enzyme method. The method comprises the following steps: the 3-amino-D-ribose generates beta-aminoribose-1-phosphate under the action of methyl thioribose kinase and ATP; the 3-amino-N, N-dimethyl adenosine is generated by the beta-amino ribose-1-phosphate and the N, N-dimethyl purine under the action of purine nucleoside phosphorylase; the puromycin is generated by the action of 3-amino-N, N-dimethyl adenosine and methyl tyrosine under the action of amino acid ligase and ATP. The invention has the following technical effects:

aiming at the limitation of fermentation and chemical preparation methods of puromycin, the invention develops an enzymatic preparation process of puromycin. Compared with the traditional route, the process has the advantages of short route, high conversion rate, mild reaction conditions, environmental protection and the like, so that the production cost of puromycin is remarkably reduced, and the safety and green index in industrial production are improved.

Drawings

FIG. 1 shows a route for the preparation of puromycin by a multi-step enzymatic process according to the present invention;

as shown in FIG. 1, the route uses 3-amino-D-ribose as a raw material, phosphorylates it with kinase (kinase) to obtain aminoribose-1-phosphate, then generates 3-amino-N, N-dimethyladenosine analogue with Purine Nucleoside Phosphorylase (PNP), and finally generates puromycin with amino acid ligase (aaLigase).

Detailed Description

The invention discloses a method for preparing puromycin by an enzyme method, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Information relating to the enzyme:

methyl thioribokinase (Kinase): derived from Bacillus subtilis (Uniprot ID: O31663, EC 2.7.1.100);

purine nucleoside phosphorylase PNP (PNP-a & PNP-b) is a purine nucleoside phosphorylase 2 engineered in Escherichia coli (Escherichia coli) (Uniprot ID: P45563, EC 2.4.2.1);

amino acid ligase aaLigase (aalignase-1 & aalignase-2): is obtained by mutating a catalytic substrate of a wide range of L-amino acid ligase in Pseudomonas syringae (UniprotID: Q842E2, EC 6.3.2.28);

acetic acid kinase AK: derived from Thermotoga maritima (Thermotoga maritima) (Unit ID: Q9WYB1, EC 2.7.2.1).

The amino acid and DNA sequences of the enzyme are as follows:

and (3) fermentation production of enzyme:

the enzyme required by the patent is prepared by constructing a corresponding gene synthesized by a company on a specific expression plasmid and then fermenting and producing the specific expression plasmid through escherichia coli. The method specifically comprises the following steps:

the genes corresponding to the above enzymes were subjected to sequence optimization, synthesized by general biology company (Chuzhou, Anhui), introduced with NdeI/XhoI cleavage sites, and subcloned into pET 28a expression vector. Transferring the plasmid with the correct sequence into E.coli (BL21) competent cells for plate culture (organisms of the Populus family) and monoclonal small-amount liquid culture, and finally performing step-by-step amplified liquid culture on the bacteria with the correct protein expression. The method specifically comprises the steps of transferring a single colony into 5mL LB culture solution (37 ℃) containing 50 mu M kanamycin for culture, inoculating the single colony into 250mL LB culture solution containing the same antibiotics after the cells grow to the logarithmic phase, transferring the single colony into a 5L culture fermentation tank for culture when the cells grow to the logarithmic phase, and finally expressing the protein. In 5L fermenter culture, 0.5mM isopropyl-beta-D-thiogalactopyranoside (IPTG) was added when the OD of the cells was about 20 to induce protein expression for 6 hours at 25 ℃ and finally cells were harvested by high speed centrifugation (4000rpm, 20min) to obtain 30-65g of enzyme-overexpressed wet cells. Taking a small amount of cells, uniformly mixing the cells with tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) buffer solution (50mM, pH 8.0) on an ice basin, then crushing the cells by using a freeze-thaw method, centrifuging at a high speed to remove cell walls, and determining protein expression by running SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) on clear liquid. The bacterial cells with correct protein expression are used for carrying out the next catalytic experiment. Specifically, the remaining cells are mixed with Tris.HCl buffer (50mM, pH 8.0) at low temperature (about 10 g of wet cells: 200mL of buffer), then the cell walls are crushed at low temperature and high pressure, and the cell walls are removed by high speed centrifugation (16000rpm, 45min) to obtain enzyme-containing clear liquid for later use (the obtained enzyme activity is 100-220U/mL, U is the enzyme amount required for converting 1 mu mol of substrate at room temperature for one minute). The LB medium is composed of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate and 5% glycerol.

The starting materials or reagents used in the present invention are commercially available.

The invention is further illustrated by the following examples:

example 1: preparation of aqueous solution of acetyl phosphate

135mL of phosphoric acid (85%, 2.0mol) was dissolved in 1.2L of ethyl acetate and then cooled to 0 ℃; to this solution 376mL of cooled acetic anhydride (4.0mol) was slowly added dropwise. The mixture was stirred at 0 ℃ for 6 hours and poured into a 5l reaction flask containing 1l water, 500 g ice and 168 g sodium bicarbonate. The mixture was continued to be stirred at low temperature until no bubbles were generated. The upper layer of ethyl acetate phase was separated and discarded, and the remaining aqueous phase was adjusted to about 3 pH and extracted twice with 2.0L, 1.0L ethyl acetate to remove most of the residual acetic acid. Finally, the pH value of the aqueous solution containing the acetyl phosphate is adjusted to be neutral by sodium hydroxide for later use, and the 1.5L of 1.2N aqueous solution of the acetyl phosphate is obtained through enzyme activity test.

Example 2: one-pot method for preparing puromycin by using liquid enzyme (Kinase, PNP-a, aalignase-1, AK)

To 1L of 100mM Tris-hydrochloric acid (Tris.HCl) pH 8.0 was added 14.9 g of 3-amino-D-ribose (100mM), 16.2 g of N, N-dimethylpurine nicotinic acid (100mM), 19.5 g of methyl tyrosine, 0.95 g of MgCl₂(10mM), 0.79 g of adenosine monosodium triphosphate (1.5mM), 170mL of the 1.2N aqueous acetyl phosphate solution prepared above (200mM) and 100mL of isopropanol (substrate cosolvent). Before adding the enzyme, the pH of the solution is adjusted to 8.0, then 1500U kinase, 1500U UPNP-a, 800U aalignase-1 and 3000U AK are added to start the enzyme reaction, the reaction solution is maintained at pH 7.0-8.5 in the reaction process, the pH of the solution is acidified to 3.0 after stirring for 4 hours at room temperature to denature and precipitate the enzyme in the reaction solution, then the pH value is adjusted back to 7.0, and then separation and purification are carried out on a nonpolar column Seplite D101 (Xian Lang New science and technology materials Co., Ltd.), and a puromycin crude product is further crystallized in ethanol/water (3:2, v: v) to obtain 29.2 g of light yellow solid (the total yield is 62%).

Example 3: one-pot method for preparing puromycin by using liquid enzyme (Kinase, PNP-b, aalignase-2, AK)

Similarly to example 2, example 3 utilizes PNP-b and aalignase-2 instead of PNP-a and aalignase-1.

To 1L of 100mM Tris-hydrochloric acid (Tris.HCl) pH 8.0 was added 7.45 g of 3-amino-D-ribose (50mM), 8.1 g of N, N-dimethylpurine nicotinic acid (50mM), 9.75 g of methyl tyrosine, 0.95 g of MgCl₂(10mM), 0.79 g of adenosine monosodium triphosphate (1.5mM), 85mL of the 1.2N aqueous acetyl phosphate solution prepared above (100mM) and 100mL of isopropanol (substrate cosolvent). Before the enzyme was added, the pH of the solution was adjusted to 8.0, then 1000U of kinase, 800U of PNP-b, 150U were added0U aalligase-2 and 2000U AK start enzyme reaction, the reaction solution is maintained at pH 7.0-8.5 in the reaction process, the solution is stirred for 6 hours at room temperature, the pH value of the solution is acidified to 3.0 to denature and precipitate the enzyme in the reaction solution, then the pH value is adjusted back to 7.0, and the solution is separated and purified by a non-polar column Seplite D101 (New science and technology materials Co., Ltd., Xian blue), and a puromycin crude product is further crystallized in ethanol/water to obtain 15.4 g of light yellow solid (the total yield is 65%).

Example 4: one-pot method for preparing puromycin by using liquid enzyme (Kinase, PNP-b, aalignase-1, AK)

Similar to examples 2 and 3, the PNP-b, aalignase-1 with the best catalytic effect was used for subsequent catalysis.

To 1L of 100mM Tris-hydrochloric acid (Tris.HCl) pH 8.0 was added 22.4 g of 3-amino-D-ribose (150mM), 24.3 g of N, N-dimethylpurine nicotinic acid (150mM), 29.3 g of methyl tyrosine, 0.95 g of MgCl₂(10mM), 0.79 g of adenosine monosodium triphosphate (1.5mM), 255mL of the 1.2N aqueous acetyl phosphate solution prepared above (300mM) and 150mL of isopropanol (substrate cosolvent). Before adding the enzyme, the pH of the solution is adjusted to 8.0, then 1200U of kinase, 1200U of PNP-b, 1200U of aalignase-1 and 4000U of AK are added to start the enzyme reaction, the reaction solution is maintained at pH 7.0-8.5 in the reaction process, the pH of the solution is acidified to 3.0 after stirring at room temperature for 6 hours to denature and precipitate the enzyme in the reaction solution, then the pH value is adjusted back to 7.0, and then separation and purification are carried out on a nonpolar column Seplite D101 (New science and technology materials Co., Ltd.) to obtain 60 g of light yellow solid (the total yield is 82%) after the puromycin crude product is further crystallized in ethanol/water.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.