CN113881728B - Preparation method of 7-aminomethyl-7-deazaguanine (PreQ 1) - Google Patents

Abstract

The invention relates to the field of nucleoside preparation, 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 (PreQ 1) by enzyme catalysis and chemical synthesis, the invention develops a method for preparing the chemical enzyme combination, 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 the industrial production while ensuring the market competitiveness.

Description

Preparation method of 7-aminomethyl-7-deazaguanine (PreQ 1)

Technical Field

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

Background

7-aminomethyl-7-deazaguanine (PreQ 1) is an important starting material for the synthesis of Q nucleosides (queuosine), a highly modified nucleoside similar to guanidine, which are widely found in certain tRNA's of bacteria and eukaryotes and are reported to be preferentially used in t-RNA synthesis, but the intrinsic mechanism of which is not fully understood. Q nucleosides are reported to have a coenzyme effect in prokaryotes and can act potentially as probiotics (growth promoting substances) in higher animals. Q nucleoside is a novel compound produced by microorganism-host interaction, and cannot be synthesized by human body, and its source is mainly human symbiotic microorganism. The base of the Q nucleoside is called Q base (queuine), the main constituent of which is 7-aminomethyl-7-deazaguanine (PreQ 1). 7-aminomethyl-7-deazaguanine is an essential starting material, both in the biosynthesis and chemical preparation of Q nucleosides.

Existing methods for preparing PreQ1 include enzymatic and chemical synthesis methods:

enzymatic method: in the in vivo synthesis of preQ1, five steps of enzyme (GCHI/QueD/QueE/QueF) transformation are required to obtain preQ1 using Guanosine Triphosphate (GTP) as a starting material. Therefore, the route is long, the overall yield is low, and no scale-up preparation using this method has been reported so far.

Chemical synthesis method: there is also little research on the chemical preparation of PreQ1, but a large amount of functional group protection, deprotection, and complicated chemical transformations are required to achieve (more than 7 steps of chemical reactions are required), and the industrial preparation possibility is low.

The preparation route of the 7-aminomethyl-7-deazapurine enzyme method is too long, and an effective in-vitro enzyme catalysis process is difficult to realize; the chemical preparation of the catalyst needs to protect and deprotect a plurality of functional groups, the whole route is long, and various toxic and harmful reagents (such as pivaloyl chloride, TMSTF, cesium acetate and the like) are needed in the chemical preparation process, so that 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 public emphasis on personal safety and natural environment protection in industrial production, the green chemical industry is a necessary trend of development. Therefore, it is important to provide a method for preparing 7-aminomethyl-7-deazaguanine (PreQ 1) by chemical enzyme method.

Disclosure of Invention

In view of the above, the present invention has developed a method for preparing 7-aminomethyl-7-deazaguanine (PreQ 1) by enzyme catalysis and chemical synthesis, which not only shortens the preparation route, but also greatly improves the efficiency of each conversion step, and at the same time, 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 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 (PreQ 1), which comprises the following steps:

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

step 2: and (3) performing enzymolysis to prepare the 7-aminomethyl-7-deazapurine (PreQ 1).

In some embodiments of the present invention, step 1 employs starting materials comprising methyl formate, methyl chloroacetate, and 2, 4-diamino-6-hydroxypyrimidine.

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

step 1-1: methyl formate and methyl chloroacetate are condensed under alkaline conditions to generate an intermediate methyl 2-chloro-3-oxopropionate (compound 1);

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

step 1-3: the ester is hydrolyzed under the weak alkaline condition 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 temperature of the condensation in step 1-1 is from-10 to 10 ℃ for a period of from 1.5 to 4.0 hours;

the temperature of the condensation in the step 1-2 is 80-100 ℃ and the time is 0.5-2.0 h;

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

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

the alkalescent condition in the step 1-3 is that the pH value is 12-14.

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

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

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

The invention uses cheap industrial chemicals (methyl formate, methyl chloroacetate and 2, 4-diamino-6-hydroxy pyrimidine) as raw materials, firstly prepares 7-carboxyl-7-denitrogen guanine (CDG) through three steps of simple chemical conversion, and then converts 7-carboxyl into 7-aminomethyl with high efficiency and specificity by utilizing two steps of convenient enzyme reaction, thereby obtaining the final product 7-aminomethyl-7-denitrogen guanine (PreQ 1). The chemical-enzyme combination method utilizes the diversity of chemical catalysis and the selectivity of the enzyme method, thereby realizing the optimization of the preparation route of the PreQ1, shortening the route, improving the final yield and effectively improving the green index of the 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 chemical enzymatic route of the present invention;

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

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

FIG. 4 shows the preparation of 7-cyano-7-deazapurine (PreQ) by CDG enzyme in example 3 ₀ )；

FIG. 5 shows the preparation of 7-aminomethyl-7-deazapurine (PreQ 1) by the PreQ0 enzyme (QueF 1) in example 4;

FIG. 6 shows the preparation of 7-aminomethyl-7-deazapurine (PreQ 1) by the PreQ0 enzyme (QueF 2) in example 5;

FIG. 7 shows the direct preparation of 7-aminomethyl-7-deazapurine (PreQ 1) (QueC+QueF1) by the CDG enzyme of example 6.

Detailed Description

The invention discloses a preparation method of 7-aminomethyl-7-deazaguanine (PreQ 1), which can be realized by appropriately improving process parameters by a person skilled in the art based on the content of the present disclosure. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

Enzyme related information:

7-cyano-7-deazaguanine synthetase (QueC synthase) derived from E.coli (Uniprot ID: P77756, EC 6.3.4.20);

cyano reductase (Nitrile Reductase, queF 1) from pectobacterium carotovorum (Pectobacterium carotovorum, uniprot ID: C6DAH4, EC 1.7.1.13);

cyano reductase (Nitrile Reductase, queF 2) from Bacillus subtilis (Bacillus subtilis, uniprot ID: O31678, EC 1.7.1.13);

polyphosphate Kinase (PPK) from Rhizobium meliloti (Rhizobium meliloti, uniprot ID: Q92SA6, EC 2.7.4.1);

the phosphite oxidase (PTDH) was engineered using a phosphite dehydrogenase in Pseudomonas stutzeri (Pseudomonas stutzeri) as a template (Uniprot ID: O69054, EC 1.20.1.1).

Fermentation production of enzyme:

the enzyme required by the invention is prepared by constructing a specific expression plasmid after the company synthesizes corresponding genes and then fermenting and producing the specific expression plasmid by escherichia coli; the method specifically comprises the following steps: after sequence optimization, the genes corresponding to the enzymes are synthesized by general biological company (Chuzhou Anhui, anhui), ndeI/XhoI restriction sites are introduced and subcloned into pET 28a expression vectors. Plasmid with correct sequence was confirmed to be transferred into E.coli (BL 21) competent cells plate culture (of the species Prinsepia) and monoclonal miniculture, the bacteria with correct protein expression are finally amplified and cultured step by step. Specifically, the single colony is transferred into 5ml LB culture solution (37 ℃) containing 50 mu M kanamycin for culture, and when the cell grows to the logarithmic phase, the cell is inoculated into 250ml LB culture solution containing the same antibiotics, and when the cell grows to the logarithmic phase, the cell is transferred into a 5L culture fermentation tank for culture, and the final protein expression is carried out. In 5L fermenter culture, 0.5mM isopropyl-beta-D-thiogalactopyranoside (IPTG) is added at 25 ℃ to induce protein expression for 6 hours when the cells OD-20, and finally, 40-70g of wet cells with over-expressed enzyme are obtained by collecting the cells (4000 rpm,20 min) by high-speed centrifugation. A small amount of cells are firstly mixed with a buffer solution (50 mM, pH 8.0) of tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) on an ice basin uniformly, then the cells are broken by a freeze thawing method, and clear liquid is subjected to SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) after cell walls are removed by high-speed centrifugation to determine protein expression. Cells with correct protein expression were used for the next catalytic experiment, specifically, the remaining cells were mixed with Tris.HCl buffer (50 mM, pH 8.0) at low temperature (200 ml buffer mixing with 10g wet cells), then crushed cell walls at low temperature Gao Yapo, and the cell walls were removed by high speed centrifugation (16000 rpm,45 min) to obtain enzyme-containing supernatant (the enzyme activity obtained was 150-300U/ml, U was the amount of enzyme required for converting 1. Mu. Mol of substrate in one minute at room temperature). LB medium consisted of: 1% tryptone, 0.5% yeast powder, 1% NaCl,1% dipotassium hydrogen phosphate and 5% glycerol.

TABLE 1

TABLE 2

The chemical enzyme method of the invention has the route shown in figure 1:

firstly, methyl formate and methyl chloroacetate are condensed under alkaline condition to generate 2-chloro-3-oxopropionate (compound 1), then the intermediate is condensed with 2, 4-diamino-6-hydroxypyrimidine in aqueous solution to form 7-methyl formate-7-deazaguanine (compound 2), and under alkaline condition, 7-carboxyl-7-deazaguanine (CDG) is obtained by ester hydrolysis; finally, 7-carboxyl-7-deazaguanine is catalyzed by 7-cyano-7-deazaguanine synthetase (EC 6.3.4.20) to generate 7-cyano-7-deazaguanine (Pre Q0), and the Pre Q0 is prepared into a final compound 7-aminomethyl-7-deazaguanine (PreQ 1) under the action of specific cyano reductase (Nitrile Reductase, queF, EC 1.7.1.13). Since Adenosine Triphosphate (ATP) and Nicotinamide Adenine Dinucleotide Phosphate (NADPH) are required for the last two-step enzyme reaction, timely regeneration of the two coenzymes can further reduce the production cost and improve the product quality.

With public emphasis on personal safety and natural environment protection in industrial production, the green chemical industry is a necessary trend of development. Considering that a single preparation method cannot achieve efficient preparation of 7-aminomethyl-7-deazapurine, the unique advantages of both transformations by combining the use of chemo-enzymes are an important option; meanwhile, due to the environment compatibility of enzyme catalysis, waste generation in the preparation process can be greatly reduced. The preparation method combines and utilizes the preQ1 chemistry and enzyme preparation process, and forms an optimal preparation route through effective optimization and integration, and the route not only greatly reduces the production cost, but also is more suitable for large-scale industrial catalysis.

In the preparation method of 7-aminomethyl-7-deazaguanine (PreQ 1), the raw materials and reagents used in the preparation method 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 roadmap is shown in fig. 2.

21.2g (350 mmol) of methyl formate are dissolved in 200ml of anhydrous Tetrahydrofuran (THF) and then cooled with stirring under ice bath; 4.32g (80 mmol) of sodium methoxide was added in portions at 4℃and stirred for 30 minutes after the addition was completed, followed by slow dropwise addition of 6.52g (60 mmol) of methyl chloroacetate using a dropping funnel. After the completion of the dropwise addition, the mixture was stirred at 4℃for 2 hours, and then the mixture was slowly heated to 25℃and stirred for 1 hour. And finally adding ice water into the reaction system to quench unreacted sodium methoxide, decompressing and pumping out the low-boiling point unreacted raw material methyl formate and solvent THF from the mixed solution by using a rotary evaporator, wherein the rest aqueous solution is the aqueous solution of the product methyl 2-chloro-3-oxopropionate (compound 1), and the compound can be directly subjected to the next conversion without separation. The specific procedure was to heat the aqueous solution to 90℃and then add 3.6g (30 mmol) of 2, 4-diamino-6-hydroxypyrimidine thereto, heat and maintain 90℃under stirring for 30 minutes, and then to filter and dry the solid formed in the solution to give 5.55g of 7-methyl formate-7-deazaguanine (compound 2) as a white solid (total yield 89%).

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

The roadmap is shown in fig. 3.

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

Example 3: preparation of 7-cyano-7-deazapurine from CDG enzyme (PreQ ₀ )

The roadmap is shown in fig. 4.

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

Example 4: preparation of 7-aminomethyl-7-deazapurine from PreQ0 enzyme (QueF 1) (PreQ 1)

The roadmap is shown in fig. 5.

To 1000ml of a solution containing 50mM Tris-HCl pH 8.0 was added 17.5g of 7-cyano-7-deazaguanurine (100 mM), 1.5g of beta-Nicotinamide Adenine Dinucleotide Phosphate (NADP) ⁺ ) Monosodium salt (0.2 mM), 52g sodium phosphite pentahydrate (240 mM) and 200ml isopropanol. After adjusting the pH of the reaction solution to 8.0, 2000U of QueF1 and 4000U of PTDH (27-67 mg) were added to start the reaction, the reaction solution was slowly stirred at room temperature for 4 hours, and the pH of the system was maintained between 7.0 and 8.5 by adding HCl or NaOH aqueous solution during the reaction. After the reaction, HCl solution was added to precipitate proteins and the resulting solution was removed by centrifugation at high speed, and finally, the resulting solution was concentrated by desalting with a reverse osmosis membrane and crystallized with ethanol/water to obtain 15.6g of a white solid (yield: 89%).

Example 5: preparation of 7-aminomethyl-7-deazapurine from PreQ0 enzyme (QueF 2) (PreQ 1)

The roadmap is shown in fig. 6.

Similar to example 4 above, this time a QueF2 enzyme was used instead of a QueF1 enzyme. Likewise, 4.37g of 7-cyano-7-deazaguanurine (50 mM), 0.75g of β -nicotinoyl are added successively to 500ml of a solution containing 50mM of tris (hydroxymethyl) aminomethane hydrochloride (Tris. HCl) pH 8.0Amine adenine dinucleotide phosphate (NADP) ⁺ ) Monosodium salt (0.2 mM), 13g sodium phosphite pentahydrate (120 mM) and 100ml isopropanol. After adjusting the pH of the reaction solution to 8.0, 3000U of QueF2 (10-20 mg) and 4000U of PTDH are added to start the reaction (PTDH 27-67 mg), the reaction solution is slowly stirred at room temperature for 8 hours, and the pH of the system is maintained between 7.0 and 8.5 by adding HCl or NaOH aqueous solution during the reaction. After the reaction, HCl solution was added to precipitate the protein, followed by high-speed centrifugation to remove the protein, and finally, the solution was concentrated by desalting with a reverse osmosis membrane, and then, crystals were formed with ethanol/water to obtain 2.7g of a white solid (yield: 61%).

Example 6: preparation of 7-aminomethyl-7-deazapurine (PreQ 1) from CDG direct enzyme (QueC+QueF1)

The roadmap is shown in fig. 7.

To 500ml of a solution containing 100mM Tris-HCl (pH 8.0) were added 4.9g of 7-methanoic acid-7-deazaguanine (CDG, 50 mM), 1.6g of amine chloride (60 mM), 0.7g of adenosine disodium salt ATP (2.5 mM), 4.2g of sodium metaphosphate (13.8 mM), 0.48g of magnesium chloride (10 mM), 0.75g of potassium chloride (20 mM), 0.75g of beta-Nicotinamide Adenine Dinucleotide Phosphate (NADP) ⁺ ) Monosodium salt (0.2 mM), 13g sodium phosphite pentahydrate (60 mM) 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), 1000U of QueF1 (3.3-6.7 mg), 2000U of polyphosphate kinase PPK (6.7-10 mg) and 3000U of PTDH (20-50 mg) are added into the solution to start the reaction, the reaction solution is slowly stirred for 6 hours at room temperature, and meanwhile, HCl or NaOH dilute aqueous solution is added at intervals in the reaction process to maintain the pH value of the system between 7.0 and 8.5. As the post-treatment of the enzyme reaction is the same, protein is precipitated by adding a dilute hydrochloric acid aqueous solution after the reaction is finished and is removed by high-speed centrifugation, a reverse osmosis membrane is utilized for desalting after the pH value of the solution is adjusted back to be neutral, D201 anion exchange resin is utilized for removing phosphoric acid impurities, and finally the obtained 7-aminomethyl-7-deazapurine crude aqueous solution is concentrated and then is crystallized by ethanol/water to obtain 4.1g of white solid (the yield of two steps is 91%).

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

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<120> preparation method of 7-aminomethyl-7-deazaguanine (PreQ 1)

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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 (5)

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

   step 1: preparing 7-formic acid-7-deazaguanine;

   step 2: the 7-aminomethyl-7-deazaguanine is prepared through enzymolysis;

   the raw materials adopted in the step 1 comprise methyl formate, methyl chloroacetate and 2, 4-diamino-6-hydroxypyrimidine;

   the step 1 specifically comprises the following steps:

   step 1-1: methyl formate and methyl chloroacetate are condensed under alkaline conditions to generate an intermediate methyl 2-chloro-3-oxopropionate;

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

   step 1-3: ester hydrolysis under weak alkaline condition to obtain 7-formic acid-7-deazaguanine;

   the enzymes in the step 2 are 7-cyano-7-deazaguanine synthetase, cyano reductase, polyphosphate kinase and phosphite oxidase;

   the reaction raw materials for enzymolysis are adenosine triphosphate and nicotinamide adenine dinucleotide phosphate;

   the amino acid sequence of the 7-cyano-7-deazaguanine synthetase is shown as SEQ ID No. 1;

   the amino acid sequences of the cyano reductase are shown as SEQ ID No.2 and SEQ ID No. 3;

   the amino acid sequence of the polyphosphate kinase is shown as SEQ ID No. 4;

   the amino acid sequence of the phosphite oxidase is shown as SEQ ID No. 5.
2. 2. The process according to claim 1, 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).
3. 3. The method according to claim 2, wherein the temperature of the condensation in step 1-1 is-10 to 10 ℃ for 1.5 to 4.0 hours;

   the temperature of the condensation in the step 1-2 is 80-100 ℃ and the time is 0.5-2 h;

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

   the alkalescent condition in the step 1-3 is that the pH value is 12-14.
5. 5. The process according to claim 4, 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.