CN113881728A - Preparation method of 7-aminomethyl-7-deazaguanine (PreQ1) - Google Patents

Abstract

The invention relates to the field of preparation of nucleosides, in particular to a preparation method of 7-aminomethyl-7-deazaguanine (PreQ 1). Aiming at the advantages and defects of the preparation method of 7-aminomethyl-7-deazaguanine (PreQ1) enzyme catalysis and chemical synthesis, the invention develops the method of chemical enzyme combination preparation, which shortens the preparation route, greatly improves the conversion efficiency of each step, and greatly reduces the waste discharge of the whole preparation route due to the participation of enzyme catalysis, thereby improving the safety and green index in industrial production while ensuring the market competitiveness.

Description

Preparation method of 7-aminomethyl-7-deazaguanine (PreQ1)

Technical Field

The invention relates to the field of preparation of nucleosides, in particular to a preparation method of 7-aminomethyl-7-deazaguanine (PreQ 1).

Background

7-aminomethyl-7-deazaguanine (PreQ1) is an important starting material for the synthesis of Q nucleoside (queosine), a highly modified nucleoside similar to guanidine, which is widely present in certain tRNAs of bacteria and eukaryotes and reported to be preferentially used in t-RNA synthesis, but the mechanism within which is not fully understood. Q nucleosides are reported to have a coenzyme effect in prokaryotes and to have a potential role as probiotics (growth promoting substances) in higher animals. The Q nucleoside is a novel compound produced by microbial-host interaction, which is not synthesized by the human body itself, and is mainly derived from human symbiotic microorganisms. The base of the Q nucleoside is called the Q base (queine), and the main constituent of the base is 7-aminomethyl-7-deazaguanine (PreQ 1). 7-aminomethyl-7-deazaguanine is an essential starting material for Q nucleosides, whether in their biosynthesis or chemical preparation.

The existing preparation methods of PreQ1 include enzymatic and chemical synthesis methods:

an enzyme method comprises the following steps: in vivo synthesis of PreQ1, using Guanosine Triphosphate (GTP) as a starting material, five steps of enzymatic conversion (GCHI/QueD/QueE/QueC/QueF) were required to obtain PreQ 1. Therefore, the route is long, the overall yield is low, and the method has not been reported to be used for scale-up preparation.

Chemical synthesis method: the chemical preparation of PreQ1 has been studied only a little, but it requires a large amount of functional group protection, deprotection, and complicated chemical transformation (more than 7 chemical reactions), and its industrial preparation is not possible.

The preparation route of the 7-aminomethyl-7-denitrified guanine enzyme method is too long, and an effective in-vitro enzyme catalysis process is difficult to realize; the chemical preparation needs to protect and remove protection on a plurality of functional groups, the whole route is long, and a plurality of toxic and harmful reagents (such as pivaloyl chloride, TMSTf, cesium acetate and the like) need to be used in the chemical preparation process, so the safety coefficient in the production process is low, the environmental compatibility is low, and the whole yield is not high; 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. Therefore, the preparation of 7-aminomethyl-7-deazaguanine (PreQ1) by a chemical enzyme method has important practical significance.

Disclosure of Invention

In view of the above, the invention develops a method for preparing by combining chemical enzymes aiming at the advantages and the defects of a preparation method of 7-aminomethyl-7-deazaguanine (PreQ1) through enzyme catalysis and chemical synthesis, which not only shortens the preparation route, but also greatly improves the conversion efficiency of each step, and simultaneously, due to the participation of enzyme catalysis, the waste emission of the whole preparation route is greatly reduced, thereby improving the safety and the green index in the industrial production while ensuring the market competitiveness.

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

the invention provides a preparation method of 7-aminomethyl-7-deazaguanine (PreQ1), which comprises the following steps:

step 1: preparing and obtaining 7-carboxyl-7-deazaguanine (CDG);

step 2: and performing enzymolysis to obtain the 7-aminomethyl-7-deazaguanine (PreQ 1).

In some embodiments of the invention, the starting materials employed in step 1 include methyl formate, methyl chloroacetate, and 2, 4-diamino-6-hydroxypyrimidine.

In some embodiments of the invention, step 1 is specifically:

step 1-1: condensing methyl formate and methyl chloroacetate under alkaline conditions to generate an intermediate 2-chloro-3-oxopropionic acid methyl ester (compound 1);

step 1-2: condensing the intermediate with 2, 4-diamino-6-hydroxypyrimidine in water solution to form a ring to obtain 7-methyl formate-7-deazaguanine (compound 2);

step 1-3: hydrolyzing the ester under alkalescent conditions to obtain 7-carboxyl-7-deazaguanine (CDG).

In some embodiments of the invention, the molar ratio of methyl formate, methyl chloroacetate to 2, 4-diamino-6-hydroxypyrimidine is: (10-12): (1.5-2.5): (0.9-1.2).

Preferably, the molar ratio of methyl formate, methyl chloroacetate to 2, 4-diamino-6-hydroxypyrimidine is: 35:6:3.

In some embodiments of the invention, the condensation in step 1-1 is carried out at a temperature of-10 to 10 ℃ for 1.5 to 4.0 hours;

the condensation in the step 1-2 is carried out at the temperature of 80-100 ℃ for 0.5-2.0 h;

in the step 1-3, the ester hydrolysis temperature is 90-120 ℃, and the time is 2-4 h.

In some embodiments of the invention, the basic conditions in step 1-1 are provided by sodium methoxide;

the weak alkaline condition in the step 1-3 is a pH value of 12-14.

In some embodiments of the invention, the enzyme activity of the 7-cyano-7-deazaguanine synthetase is 75-120U/mg, and the enzyme activity of the cyano reductase is 150-300U/mg.

In some embodiments of the invention, one or both of the polyphosphate kinases PPK or PTDH are also included.

In some embodiments of the invention, the enzymatic hydrolysis further comprises reaction raw materials of Adenosine Triphosphate (ATP) and Nicotinamide Adenine Dinucleotide Phosphate (NADPH).

The invention uses cheap industrial chemicals (methyl formate, methyl chloroacetate and 2, 4-diamino-6-hydroxypyrimidine) as raw materials, prepares 7-carboxyl-7-deazaguanine (CDG) by three steps of simple chemical conversion, and then converts 7-carboxyl into 7-aminomethyl by two steps of convenient enzyme reactions in a high-efficiency and specific manner so as to obtain a final product, namely the 7-aminomethyl-7-deazaguanine (PreQ 1). The chemical-enzyme combination method utilizes the diversity of chemical catalysis and the selectivity of an enzyme method, thereby realizing the optimization of a PreQ1 preparation route, shortening the route, improving the final yield and effectively improving the green index of large-scale production.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a route for the chemoenzymatic preparation of the present invention;

FIG. 2 shows a chemical route for the preparation of 7-methyl formate-7-deazaguanine (Compound 2) in example 1;

FIG. 3 shows a chemical route for the preparation of 7-formic acid-7-deazaguanine (CDG) in example 2;

FIG. 4 shows the CDG enzyme preparation of 7-cyano-7-deazaguanine (PreQ) in example 3₀)；

FIG. 5 shows the preparation of 7-aminomethyl-7-deazaguanine (PreQ1) by the PreQ0 enzyme (QueF1) in example 4;

FIG. 6 shows the preparation of 7-aminomethyl-7-deazaguanine (PreQ1) by the PreQ0 enzyme (QueF2) in example 5;

FIG. 7 shows the direct preparation of 7-aminomethyl-7-deazaguanine (PreQ1) (QueC + QueF1) by the CDG enzyme in example 6.

Detailed Description

The invention discloses a preparation method of 7-aminomethyl-7-deazaguanine (PreQ1), which can be realized by appropriately improving process parameters by persons skilled in the art with reference to the content in the text. 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:

7-cyano-7-deazaguanine synthetase (QueC synthsase) from Escherichia coli (Unit prot ID: P77756, EC 6.3.4.20);

a cyano Reductase (Nitrile Reductase, QueF1) derived from Pectinatus carotovorus (Pectinatus carotovorum, Uniprot ID: C6DAH4, EC 1.7.1.13);

cyano Reductase (NitrileReductase, QueF2) derived from Bacillus subtilis (Uniprot ID: O31678, EC 1.7.1.13);

polyphosphate Kinase (PPK) derived from Rhizobium meliloti (Rhizobium meliloti, Unit project ID: Q92SA6, EC 2.7.4.1);

phosphonite oxidase (PTDH) is prepared by using a phosphate dehydrogenase in Pseudomonas stutzeri (Uniprot ID: O69054, EC 1.20.1.1).

And (3) fermentation production of enzyme:

the enzyme required by the invention is prepared by constructing a corresponding gene synthesized by a company on a specific expression plasmid and then fermenting and producing 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 of LB culture solution (37 ℃) containing 50 mu M kanamycin for culture, inoculating the single colony into 250ml of LB culture solution containing the same antibiotics after the cell grows to the logarithmic phase, transferring the single colony into a 5L culture fermentation tank for culture when the cell grows to the logarithmic phase, and finally expressing the protein. In 5L fermentation tank culture, when the cell OD is 20, 0.5mM isopropyl-beta-D-thiogalactopyranoside (IPTG) is added to induce protein expression for 6 hours at 25 ℃, and finally, the cells are collected by high-speed centrifugation (4000rpm,20min) to obtain 40-70g of wet cells with enzyme over-expression. 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, and specifically, the residual cells and Tris.HCl buffer (50mM, pH 8.0) are uniformly mixed at low temperature (wet cells: 200ml buffer is mixed), then cell walls are broken at low temperature and high pressure, enzyme-containing clear liquid is obtained for standby after high-speed centrifugation (16000rpm,45min) is carried out to remove the cell walls (the obtained enzyme activity is 150-300U/ml, and 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.

TABLE 1

TABLE 2

The preparation route of the chemoenzymatic method is shown in figure 1:

firstly, methyl formate and methyl chloroacetate are condensed under alkaline conditions to generate 2-chloro-3-oxopropionic acid methyl ester (compound 1), then the intermediate and 2, 4-diamino-6-hydroxypyrimidine are condensed in aqueous solution to form ring to obtain 7-methyl formate-7-deazaguanine (compound 2), and ester hydrolysis is carried out under alkalescent conditions to obtain 7-carboxyl-7-deazaguanine (CDG); finally, 7-carboxyl-7-deazaguanine is catalyzed by 7-cyano-7-deazaguanine synthetase (QueC synthsase, EC 6.3.4.20) to generate 7-cyano-7-deazaguanine (Pre Q0), and PreQ0 is prepared under the action of specific cyano Reductase (Nitril Reductase, QueF, EC 1.7.1.13) to obtain the final compound 7-aminomethyl-7-deazaguanine (PreQ 1). Because Adenosine Triphosphate (ATP) and Nicotinamide Adenine Dinucleotide Phosphate (NADPH) are needed in the last two steps of enzyme reaction, the timely regeneration of the two coenzymes can further reduce the production cost and improve the product quality.

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. Considering that a single preparation method cannot realize the efficient preparation of 7-aminomethyl-7-deazaguanine, it becomes an important choice by combining the respective unique advantages of both chemo-enzymatic conversions; meanwhile, due to the environment compatibility of enzyme catalysis, the generation of waste in the preparation process can be greatly reduced. The optimal preparation route is formed by effectively optimizing and integrating the PreQ1 chemical and enzyme preparation processes, so that the route not only greatly reduces the production cost, but also is more suitable for large-scale industrial catalysis.

In the preparation method of the 7-aminomethyl-7-deazaguanine (PreQ1), the used raw materials and reagents can be purchased from the market.

The invention is further illustrated by the following examples:

example 1: chemical preparation of 7-methyl formate-7-deazaguanine (Compound 2)

The route pattern is shown in fig. 2.

Methyl formate 21.2g (350mmol) was dissolved in 200ml of anhydrous Tetrahydrofuran (THF), followed by cooling with stirring under ice bath; after addition of 4.32g (80mmol) of sodium methoxide in portions at 4 ℃ and stirring for 30 minutes, 6.52g (60mmol) of methyl chloroacetate was slowly added dropwise from the dropping funnel. After the dropwise addition, the mixture was stirred at 4 ℃ for 2 hours, then slowly raised to 25 ℃ and stirred for 1 hour. And finally, adding ice water into a reaction system to quench unreacted sodium methoxide, decompressing the mixed solution by using a rotary evaporator to remove unreacted raw material methyl formate with low boiling point and solvent THF, wherein the residual aqueous solution is the aqueous solution of the product methyl 2-chloro-3-oxopropionate (compound 1), and the compound can be directly converted in the next step without separation. Specifically, the aqueous solution was heated to 90 ℃ and then 3.6g (30mmol) of 2, 4-diamino-6-hydroxypyrimidine was added thereto, and the mixture was stirred for 30 minutes while maintaining the temperature at 90 ℃ to obtain a solid produced in the solution, which was then filtered and dried to obtain 5.55g of 7-methyl formate-7-deazaguanine (Compound 2) as a white solid (total yield 89%).

Example 2: chemical preparation of 7-formic acid-7-deazaguanine (CDG)

The route pattern is shown in fig. 3.

10g of 7-formic acid-7-deazaguanine (48mmol) are admixed with H₂To a mixed solution of O (500ml) and DMSO (100ml), 6ml (5N) of an aqueous sodium hydroxide solution was added, and the mixture was allowed to flow at 100 ℃ for 2 hours. Finally, the solution was cooled to 25 ℃ and a white solid precipitated during this process, and the solid was filtered off (8.5g, 91% yield), which was the target compound 7-formic acid-7-deazaguanine (CDG).

Example 3: preparation of 7-cyano-7-deazaguanine (PreQ) from CDG enzyme₀)

The route pattern is shown in fig. 4.

To 500ml of a solution containing 100mM Tris-HCl (pH 8.0) followed by 9.7g 7-formic acid-7-deazaguanine (CDG, 100mM), 3.2g amine chloride (120mM), 1.4g adenosine disodium triphosphate ATP (5mM), 8.4g sodium hexametaphosphate (27.5mM), 0.48g magnesium chloride (10mM), 0.75g potassium chloride (20 mM); after the pH value is adjusted to 8.0, 1000U of 7-cyano-7-deazaguanine synthetase QueC (8.3-13.3 mg) and 1500U of polyphosphate kinase PPK (5.0-7.5 mg) are added into the solution to start reaction, in the reaction process, the pH value of a reaction system is maintained at 6.5-9.0 by adding 0.1N HCl or NaOH aqueous solution, the reaction is completed after stirring at room temperature for 5 hours, then HCl is added to acidify and precipitate reaction enzyme (the pH value of the solution is adjusted to 1.5 and the solution is rapidly stirred), protein impurities are removed by centrifugation, reverse osmosis is used for desalting after the pH value of the solution is adjusted to 7.0, finally phosphoric acid impurities are removed by using D201 anion exchange resin (deionized water is used as an eluent, 7-cyano-7-deaza guanine and resin are combined to flow out weakly and directly), and a freeze-dried crude product passes through pure water: ethanol 1:3v/v crystallization gave finally 7.4g of a white solid (84% final yield).

Example 4: preparation of 7-aminomethyl-7-deazaguanine (PreQ1) from PreQ0 enzyme (QueF1)

The route pattern is shown in fig. 5.

Likewise, to 1000ml of a solution containing 50mM Tris-hydrochloric acid (Tris.HCl) pH 8.0 was added 17.5g of 7-cyano-7-deazaguanine (100mM),1.5g of beta-Nicotinamide Adenine Dinucleotide Phosphate (NADP)⁺) Monosodium salt (0.2mM),52g sodium phosphite pentahydrate (240mM) and 200ml isopropanol. After the pH value of the reaction solution is adjusted to 8.0, 2000U of QueF1 and 4000U of PTDH (27-67 mg) are added to start the reaction, the reaction solution is slowly stirred for 4 hours at room temperature, and HCl or NaOH aqueous solution is added to maintain the pH value of the system between 7.0 and 8.5 in the reaction process. After the reaction, HCl solution was added to precipitate the protein and centrifuged at high speed, and finally, the protein was concentrated by reverse osmosis membrane to remove salt and crystallized from ethanol/water to obtain 15.6g of white solid (yield 89%).

Example 5: preparation of 7-aminomethyl-7-deazaguanine (PreQ1) from PreQ0 enzyme (QueF2)

The route pattern is shown in fig. 6.

Similar to example 4 above, this time with QueF2 enzyme instead of QueF1 enzyme. Similarly, to 500ml of a Tris-HCl solution containing 50mM of Tris-HCl pH 8.0 was added 4.37g of 7-cyano-7-deazaguanine (50mM),0.75g of β -Nicotinamide Adenine Dinucleotide Phosphate (NADP)⁺) Monosodium salt (0.2mM),13g sodium phosphite pentahydrate (120mM) and 100ml isopropanol. After the pH value of the reaction solution is adjusted to 8.0, 3000U of QueF2 (10-20 mg) and 4000U of PTDH are added to start the reaction (PTDH 27-67mg), the reaction solution is slowly stirred for 8 hours at room temperature, and the pH value of the system is maintained between 7.0 and 8.5 by adding HCl or NaOH aqueous solution in the reaction process. After the reaction, HCl solution was added to precipitate protein and the protein was removed by high speed centrifugation, and finally, after desalting concentration by reverse osmosis membrane, 2.7g of white solid was obtained by ethanol/water crystallization (yield 61%).

Example 6: enzymatic preparation of 7-aminomethyl-7-deazaguanine (PreQ1) (QueC + QueF1) directly from CDG

The route pattern is shown in fig. 7.

To 500ml of a solution containing 100mM Tris-HCl pH 8.0 was added 4.9g 7-formic acid-7-deazaguanine (CDG, 50mM), 1.6g amine chloride (60mM), 0.7g adenosine disodium triphosphate ATP (2.5mM), 4.2g sodium hexametaphosphate (13.8mM), 0.48g magnesium chloride (10mM), 0.75g potassium chloride (20mM), 0.75g beta-Nicotinamide Adenine Dinucleotide Phosphate (NADP)⁺) Monosodium salt (0.2mM),13g sodium phosphite pentahydrate (60mM) and 100ml isopropanol; after the pH value of the solution is adjusted to 8.0, 1000U of 7-cyano-7-deazaguanine synthetase QueC (8.3-13.3 mg) and 1000U of QueF1 (3.3-6) are added.7mg), 2000U of PPK (6.7-10 mg) and 3000U of PTDH (20-50 mg) were added to the solution to start the reaction, and the reaction solution was slowly stirred at room temperature for 6 hours while HCl or NaOH diluted aqueous solution was intermittently added during the reaction to maintain the pH of the system at 7.0-8.5. After the completion of the above-mentioned enzymatic reaction, the same procedure was followed as described above, and the protein was precipitated by adding dilute aqueous hydrochloric acid and removed by high-speed centrifugation, and when the pH of the solution was adjusted to neutral, the salt was removed by means of a reverse osmosis membrane, and the phosphoric acid-containing impurities were removed by means of D201 anion exchange resin, and the resulting crude aqueous solution of 7-aminomethyl-7-deazaguanine was concentrated and crystallized from ethanol/water to give 4.1g of a white solid (yield in two steps: 91%).

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.

Sequence listing

<110> Shenzhen Dardlin biotechnologies Limited

<120> preparation method of 7-aminomethyl-7-deazaguanine (PreQ1)

<130> S21P002357

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 231

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Lys Arg Ala Val Val Val Phe Ser Gly Gly Gln Asp Ser Thr Thr

1 5 10 15

Cys Leu Val Gln Ala Leu Gln Gln Tyr Asp Glu Val His Cys Val Thr

20 25 30

Phe Asp Tyr Gly Gln Arg His Arg Ala Glu Ile Asp Val Ala Arg Glu

35 40 45

Leu Ala Leu Lys Leu Gly Ala Arg Ala His Lys Val Leu Asp Val Thr

50 55 60

Leu Leu Asn Glu Leu Ala Val Ser Ser Leu Thr Arg Asp Ser Ile Pro

65 70 75 80

Val Pro Asp Tyr Glu Pro Glu Ala Asp Gly Ile Pro Asn Thr Phe Val

85 90 95

Pro Gly Arg Asn Ile Leu Phe Leu Thr Leu Ala Ala Ile Tyr Ala Tyr

100 105 110

Gln Val Lys Ala Glu Ala Val Ile Thr Gly Val Cys Glu Thr Asp Phe

115 120 125

Ser Gly Tyr Pro Asp Cys Arg Asp Glu Phe Val Lys Ala Leu Asn His

130 135 140

Ala Val Ser Leu Gly Met Ala Lys Asp Ile Arg Phe Glu Thr Pro Leu

145 150 155 160

Met Trp Ile Asp Lys Ala Glu Thr Trp Ala Leu Ala Asp Tyr Tyr Gly

165 170 175

Lys Leu Asp Leu Val Arg Asn Glu Thr Leu Thr Cys Tyr Asn Gly Phe

180 185 190

Lys Gly Asp Gly Cys Gly His Cys Ala Ala Cys Asn Leu Arg Ala Asn

195 200 205

Gly Leu Asn His Tyr Leu Ala Asp Lys Pro Thr Val Met Ala Ala Met

210 215 220

Lys Gln Lys Thr Gly Leu Arg

225 230

<210> 2

<211> 282

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ser Val Tyr Asp Lys His Gln Ala Leu Ser Gly Leu Thr Leu Gly

1 5 10 15

Lys Pro Thr Pro Tyr His Asp Arg Tyr Asp Ala Ala Leu Leu Gln Pro

20 25 30

Val Pro Arg Ser Leu Asn Arg Asp Pro Leu Gly Ile His Pro Asp Ser

35 40 45

Leu Pro Phe His Gly Ala Asp Ile Trp Thr Leu Tyr Glu Leu Ser Trp

50 55 60

Leu Asn Asn Arg Gly Val Pro Gln Val Ala Val Gly Glu Met His Leu

65 70 75 80

Asn Ala Glu Ser Leu Asn Leu Ile Glu Ser Lys Ser Phe Lys Leu Tyr

85 90 95

Leu Asn Ser Phe Asn Gln Thr Thr Phe Asp Ser Trp Glu Ser Val Arg

100 105 110

Ala Thr Leu Ala Asn Asp Leu Ala His Cys Ala Gln Gly Asp Val Ser

115 120 125

Ile Thr Leu Phe Lys Leu Ser Glu Leu Glu Gly Gln Pro Leu Ala Gly

130 135 140

Phe Thr Gly Glu Cys Ile Asp Asp Gln Asp Ile Gln Ile Asp Ser Tyr

145 150 155 160

Asp Phe Asn Ala Asp Tyr Leu Ala Thr Asn Glu Gln Asp Ala Pro Val

165 170 175

Val Glu Glu Thr Leu Val Ser His Leu Leu Lys Ser Asn Cys Leu Ile

180 185 190

Thr His Gln Pro Asp Trp Gly Ser Val Gln Ile His Tyr Arg Gly Lys

195 200 205

Arg Ile Asn Arg Glu Ala Leu Leu Arg Tyr Ile Ile Ser Phe Arg His

210 215 220

His Asn Glu Phe His Glu Gln Cys Val Glu Arg Ile Phe Asn Asp Ile

225 230 235 240

Met Arg Tyr Tyr Gln Pro Glu Lys Leu Ser Val Tyr Ala Arg Tyr Thr

245 250 255

Arg Arg Gly Gly Leu Asp Ile Asn Pro Trp Arg Ser Asn Thr Ala Phe

260 265 270

Asn Ala Pro Asn Gly Arg Leu Pro Arg Gln

275 280

<210> 3

<211> 165

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Thr Thr Arg Lys Glu Ser Glu Leu Glu Gly Val Thr Leu Leu Gly

1 5 10 15

Asn Gln Gly Thr Asn Tyr Leu Phe Glu Tyr Ala Pro Asp Val Leu Glu

20 25 30

Ser Phe Pro Asn Lys His Val Asn Arg Asp Tyr Phe Val Lys Phe Asn

35 40 45

Cys Pro Glu Phe Thr Ser Leu Cys Pro Lys Thr Gly Gln Pro Asp Phe

50 55 60

Ala Thr Ile Tyr Ile Ser Tyr Ile Pro Asp Glu Lys Met Val Glu Ser

65 70 75 80

Lys Ser Leu Lys Leu Tyr Leu Phe Ser Phe Arg Asn His Gly Asp Phe

85 90 95

His Glu Asp Cys Met Asn Ile Ile Met Asn Asp Leu Ile Glu Leu Met

100 105 110

Asp Pro Arg Tyr Ile Glu Val Trp Gly Lys Phe Thr Pro Arg Gly Gly

115 120 125

Ile Ser Ile Asp Pro Tyr Thr Asn Tyr Gly Lys Pro Gly Thr Lys Tyr

130 135 140

Glu Lys Met Ala Glu Tyr Arg Met Met Asn His Asp Leu Tyr Pro Glu

145 150 155 160

Thr Ile Asp Asn Arg

165

<210> 4

<211> 300

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ala Leu Asp Glu Ala Pro Ala Glu Ala Arg Pro Gly Ser Arg Ala

1 5 10 15

Val Glu Leu Glu Ile Asp Gly Arg Ser Arg Ile Phe Asp Ile Asp Asp

20 25 30

Pro Asp Leu Pro Lys Trp Ile Asp Glu Glu Ala Phe Arg Ser Asp Asp

35 40 45

Tyr Pro Tyr Lys Lys Lys Leu Asp Arg Glu Glu Tyr Glu Glu Thr Leu

50 55 60

Thr Lys Leu Gln Ile Glu Leu Val Lys Val Gln Phe Trp Met Gln Ala

65 70 75 80

Thr Gly Lys Arg Val Met Ala Val Phe Glu Gly Arg Asp Ala Ala Gly

85 90 95

Lys Gly Gly Ala Ile His Ala Thr Thr Ala Asn Met Asn Pro Arg Ser

100 105 110

Ala Arg Val Val Ala Leu Thr Lys Pro Thr Glu Thr Glu Arg Gly Gln

115 120 125

Trp Tyr Phe Gln Arg Tyr Val Ala Thr Phe Pro Thr Ala Gly Glu Phe

130 135 140

Val Leu Phe Asp Arg Ser Trp Tyr Asn Arg Ala Gly Val Glu Pro Val

145 150 155 160

Met Gly Phe Cys Thr Pro Asp Gln Tyr Glu Gln Phe Leu Lys Glu Ala

165 170 175

Pro Arg Phe Glu Glu Met Ile Ala Asn Glu Gly Ile His Leu Phe Lys

180 185 190

Phe Trp Ile Asn Ile Gly Arg Glu Met Gln Leu Lys Arg Phe His Asp

195 200 205

Arg Arg His Asp Pro Leu Lys Ile Trp Lys Leu Ser Pro Met Asp Ile

210 215 220

Ala Ala Leu Ser Lys Trp Asp Asp Tyr Thr Gly Lys Arg Asp Arg Met

225 230 235 240

Leu Lys Glu Thr His Thr Glu His Gly Pro Trp Ala Val Ile Arg Gly

245 250 255

Asn Asp Lys Arg Arg Ser Arg Ile Asn Val Ile Arg His Met Leu Thr

260 265 270

Lys Leu Asp Tyr Asp Gly Lys Asp Glu Ala Ala Ile Gly Glu Val Asp

275 280 285

Glu Lys Ile Leu Gly Ser Gly Pro Gly Phe Leu Arg

290 295 300

<210> 5

<211> 336

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Leu Pro Lys Leu Val Ile Thr His Arg Val His Asp Glu Ile Leu

1 5 10 15

Gln Leu Leu Ala Pro His Cys Glu Leu Met Thr Asn Gln Thr Asp Ser

20 25 30

Thr Leu Thr Arg Glu Glu Ile Leu Arg Arg Cys Arg Asp Ala Gln Ala

35 40 45

Met Met Ala Phe Met Pro Asp Arg Val Asp Ala Asp Phe Leu Gln Ala

50 55 60

Cys Pro Glu Leu Arg Val Ile Gly Cys Ala Leu Lys Gly Phe Asp Asn

65 70 75 80

Phe Asp Val Asp Ala Cys Thr Ala Arg Gly Val Trp Leu Thr Phe Val

85 90 95

Pro Asp Leu Leu Thr Val Pro Thr Ala Glu Leu Ala Ile Gly Leu Ala

100 105 110

Val Gly Leu Gly Arg His Leu Arg Ala Ala Asp Ala Phe Val Arg Ser

115 120 125

Gly Lys Phe Arg Gly Trp Gln Pro Arg Phe Tyr Gly Thr Gly Leu Asp

130 135 140

Asn Ala Thr Val Gly Phe Leu Gly Met Gly Ala Ile Gly Leu Ala Met

145 150 155 160

Ala Asp Arg Leu Gln Gly Trp Gly Ala Thr Leu Gln Tyr His Glu Arg

165 170 175

Lys Ala Leu Asp Thr Gln Thr Glu Gln Arg Leu Gly Leu Arg Gln Val

180 185 190

Ala Cys Ser Glu Leu Phe Ala Ser Ser Asp Phe Ile Leu Leu Ala Leu

195 200 205

Pro Leu Asn Ala Asp Thr Leu His Leu Val Asn Ala Glu Leu Leu Ala

210 215 220

Leu Val Arg Pro Gly Ala Leu Leu Val Asn Pro Cys Arg Gly Ser Val

225 230 235 240

Val Asp Glu Ala Ala Val Leu Ala Ala Leu Glu Arg Gly Gln Leu Gly

245 250 255

Gly Tyr Ala Ala Asp Val Phe Glu Met Glu Asp Trp Ala Arg Ala Asp

260 265 270

Arg Pro Gln Gln Ile Asp Pro Ala Leu Leu Ala His Pro Asn Thr Leu

275 280 285

Phe Thr Pro His Ile Gly Ser Ala Val Arg Ala Val Arg Leu Glu Ile

290 295 300

Glu Arg Cys Ala Ala Gln Asn Ile Leu Gln Ala Leu Ala Gly Glu Arg

305 310 315 320

Pro Ile Asn Ala Val Asn Arg Leu Pro Lys Ala Glu Pro Ala Ala Cys

325 330 335

<210> 6

<211> 696

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgaaacgtg ctgtcgttgt gttcagtgga ggtcaggatt ccaccacctg tctggtgcag 60

gcattacaac aatatgatga agtccattgc gtgacgttcg attacggtca gcggcatcgc 120

gcagaaatcg acgtggcacg cgaactggcg ctgaaactgg gggcacgcgc gcataaggtg 180

ctggatgtca ccctgctcaa cgagctggcg gtcagtagcc tgacgcgtga cagcattccg 240

gtgcctgatt atgaacctga agccgatggt atcccgaata cgtttgtccc agggcgtaat 300

attttgttcc tgacgctggc ggcaatatat gcgtatcagg taaaagcaga agccgtaatt 360

actggcgtct gcgaaacgga tttctccggc tacccggatt gccgcgatga gtttgtgaaa 420

gcactaaacc atgccgtcag tttgggcatg gcgaaagata ttcgttttga aacgccgctg 480

atgtggattg ataaagcgga aacctgggcg ctggcagatt attacggcaa actggattta 540

gtccgtaacg aaacgttgac ctgctataac ggctttaaag gcgacggttg cggtcattgt 600

gcggcatgta atttacgcgc caacggtttg aatcattatc tggccgataa accgacggtg 660

atggcagcga tgaagcagaa aaccgggttg aggtaa 696

<210> 7

<211> 849

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgtccgttt atgacaagca ccaggccctg agcgggctga cattgggcaa acccactccc 60

taccacgacc gctatgatgc cgcccttctg caacccgtgc cacgtagcct gaaccgcgat 120

ccactcggca ttcatcctga tagcctacct tttcatggcg cagatatctg gacgctctac 180

gagctttcct ggctgaacaa ccgtggcgtg cctcaggtag ccgtcggtga aatgcatctc 240

aatgcggaaa gcctgaatct gattgaatca aaaagtttta agctgtacct gaacagcttt 300

aatcagacga cattcgacag ttgggagagc gtacgcgcga cgttagccaa cgacctggcg 360

cactgtgcac agggggacgt cagcatcacg cttttcaaac tcagcgagct cgaaggccag 420

ccgctagcgg gattcactgg cgaatgcatc gacgatcaag acattcagat cgacagctac 480

gacttcaacg ccgactatct ggcgacaaac gaacaggacg cgcctgtcgt tgaagaaacg 540

ctggtcagcc acctgctgaa atccaactgt ttgatcaccc atcagcccga ctggggctct 600

gtacagatcc actatcgcgg caaacgcatt aaccgtgaag cactgctgcg ctacattatc 660

tcgtttcgtc atcataacga atttcatgaa cagtgtgtgg aacgaatttt taacgacatc 720

atgcgctact accagccgga aaaactcagc gtttacgccc gctatacccg acgcggcggg 780

ctggacatca acccgtggcg cagcaatacc gcgtttaacg caccaaatgg acgcctgccg 840

cgtcagtaa 849

<210> 8

<211> 498

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgacgacaa gaaaagaatc agaattagaa ggtgtaacat tgctaggcaa tcaaggtaca 60

aattatttgt tcgaatatgc accggacgtg ctggaatcct tccctaataa acatgtaaac 120

cgtgattact ttgtaaaatt caattgcccg gaattcacat ctttatgtcc taaaacaggc 180

cagcctgact ttgcgacaat ctacatcagc tacattcctg atgaaaaaat ggttgaaagc 240

aaatcattaa agctgtatct attcagcttc agaaaccatg gtgacttcca cgaggactgc 300

atgaatatca tcatgaacga cttgattgaa ttaatggacc cgcgctacat tgaagtatgg 360

ggcaaattca cgccaagagg cggaatttcc attgatccgt acacaaacta cggaaagcct 420

ggcacgaagt atgagaaaat ggccgaatac cgtatgatga accatgattt gtatccggag 480

acaattgata atcgttaa 498

<210> 9

<211> 903

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atggcactcg acgaagcacc ggccgaagca aggccgggga gccgggcggt cgaactggag 60

atcgacggca gaagccgcat cttcgacatc gacgatccgg acctgccgaa atggatcgac 120

gaggaggcct tccgctccga cgattacccc tacaagaaaa aactcgatcg ggaggaatac 180

gaagaaacgc tgacgaagct gcagatcgaa ctggtcaagg tccagttctg gatgcaggcg 240

accggcaagc gcgtgatggc ggtcttcgag ggacgcgacg ctgccggcaa gggtggtgcg 300

atccacgcga cgacggccaa tatgaacccc cgctccgcgc gcgtcgtcgc actgacgaaa 360

ccgacggaga ccgaacgggg ccagtggtac ttccagcgct atgtcgcaac cttcccgacc 420

gccggcgagt tcgtcctttt cgaccgctcc tggtacaacc gcgccggtgt cgaaccggtc 480

atgggctttt gcacccccga ccagtacgag caattcctta aagaggcgcc ccgcttcgag 540

gagatgatcg cgaacgaggg catccatctc ttcaagtttt ggatcaatat cggccgggaa 600

atgcaactga agcgcttcca tgaccggcgc cacgatccgt tgaagatctg gaagctttcg 660

ccgatggaca tcgcggcgct gagcaagtgg gacgactaca ccggaaaacg cgaccgtatg 720

ctgaaggaaa cgcacacgga gcacgggcca tgggcggtca tccgcggcaa cgacaagcgc 780

cgctcgcgga tcaacgtgat ccgccacatg ctgacgaagc tcgactatga cggcaaggac 840

gaggcggcga tcggagaggt cgacgaaaag atcctcggct ccggccccgg ttttctcagg 900

tga 903

<210> 10

<211> 1011

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgttaccga aattagttat cacgcacaga gtgcacgacg aaatccttca attgctggcc 60

cctcattgtg agttgatgac caaccaaacc gattctaccc tgacgagaga agagatactg 120

cgccgttgca gagacgcaca agccatgatg gcgtttatgc cggaccgtgt agatgcagac 180

tttcttcaag cttgcccgga acttcgggtc attggttgtg ctttgaaagg gttcgacaac 240

tttgacgtgg atgcgtgtac tgcacgcggg gtatggctta cttttgtacc tgacttattg 300

acggttccca ctgccgagct tgctattggc ctggccgtcg gattaggccg ccatttacgt 360

gcggcagatg cgttcgtacg gagtgggaag tttcggggct ggcaaccgcg attctacggg 420

actggattgg ataacgccac tgtaggtttc cttgggatgg gtgccatagg tttagctatg 480

gcagatagat tacaggggtg gggagctacc cttcaatatc atgagcgtaa agcattggat 540

acacaaacag aacagcgctt gggtcttaga caggtcgcgt gctcggaact tttcgcttcc 600

tcagacttca tactgttggc cttgccactt aacgctgaca ctctacattt ggtaaacgct 660

gaattgctgg ctttggtacg tcccggcgca ctgttagtta atccgtgccg gggctcggtg 720

gtagacgagg cagccgtgct ggcagcgctt gagagagggc aacttggcgg atatgctgca 780

gacgtgttcg agatggaaga ctgggcccgc gcggaccgtc cacagcaaat cgatcctgcg 840

ttgttggccc accctaatac tttatttact ccgcacatcg gatcagcggt gagagcggtg 900

cggcttgaga ttgagcgttg cgcagctcag aacatcctcc aggcgctggc aggagaacgt 960

ccaattaatg ctgtaaatcg tttaccgaag gctgaaccag cagcttgttg a 1011

Claims (10)

1. A preparation method of 1.7-aminomethyl-7-deazaguanine is characterized by comprising the following steps:

   step 1: preparing and obtaining 7-carboxyl-7-deazaguanine;

   step 2: and performing enzymolysis to obtain the 7-aminomethyl-7-denitrified guanine.
2. 2. The method according to claim 1, wherein the starting materials used in step 1 comprise methyl formate, methyl chloroacetate and 2, 4-diamino-6-hydroxypyrimidine.
3. 3. The method according to claim 1 or 2, wherein step 1 is specifically:

   step 1-1: condensing methyl formate and methyl chloroacetate under an alkaline condition to generate an intermediate 2-chloro-3-oxopropionic acid methyl ester;

   step 1-2: condensing the intermediate with 2, 4-diamino-6-hydroxypyrimidine in an aqueous solution to form a ring to obtain 7-methyl formate-7-deazaguanine;

   step 1-3: hydrolyzing the ester under alkalescent conditions to obtain the 7-carboxyl-7-deazaguanine.
4. 4. The process according to claim 3, wherein the molar ratio of methyl formate, methyl chloroacetate to 2, 4-diamino-6-hydroxypyrimidine is: (10-12): (1.5-2.5): (0.9-1.2).
5. 5. The method according to claim 3 or 4, wherein the condensation in step 1-1 is carried out at a temperature of-10 to 10 ℃ for 1.5 to 4.0 hours;

   the condensation in the step 1-2 is carried out at the temperature of 80-100 ℃ for 0.5-2 h;

   in the step 1-3, the ester hydrolysis temperature is 90-120 ℃, and the time is 2-4 h.
6. 6. The process according to claim 3 or 4, wherein the basic conditions in step 1-1 are provided by sodium methoxide;

   the weak alkaline condition in the step 1-3 is a pH value of 12-14.
7. 7. The method according to any one of claims 1 to 6, wherein the enzyme in step 2 comprises 7-cyano-7-deazaguanine synthetase and/or cyano reductase.
8. 8. The method according to claim 7, wherein the enzyme activity of the 7-cyano-7-deazaguanine synthase is 75 to 120U/mg, and the enzyme activity of the cyano reductase is 150 to 300U/mg.
9. 9. The method of claim 8, further comprising one or both of the polyphosphate kinases PPK and PTDH.
10. 10. The method of any one of claims 1 to 9, wherein the enzymatic hydrolysis further comprises reacting raw materials of adenosine triphosphate and nicotinamide adenine dinucleotide phosphate.

Images