KR101818564B1 - Methods for Preparing 3'-amino-2',3'-dideoxyguanosine by Using Nucleoside Phosphorylases Derived from Bacillus and Adenosine Deaminase Derived from Lactococcus - Google Patents

Abstract

The present invention relates to a process for preparing 3'-amino-2 ', 3'-dideoxyguanosine using bacillus stearothermophilus-derived nucleoside phosphorylase and lactococcus lactis-derived adenosine deaminase.
According to the method for producing 3'-amino-2 ', 3'-dideoxy guanosine according to the present invention, 3'-amino-2', 3'-dideoxy guanosine is converted and purified at high yield and high purity, 3'-amino-2 ', 3'-dideoxy guanosine can be mass-produced efficiently.

Description

Amino-2 ', 3'-dideoxyguanosine using Bacillus-derived nucleoside phosphorylase and Lactococcus-derived adenosine deaminase (Methods for Preparing 3'-amino-2', 3 '-dideoxyguanosine by Nucleoside Phosphorylases Derived from Bacillus and Adenosine Deaminase Derived from Lactococcus}

The present invention relates to a process for the preparation of 3'-amino-2 ', 3'-dideoxyguanosine using Bacillus-derived nucleoside phosphorylase and Lactococcus-derived adenosine deaminase.

Extensive research is being conducted on the use of oligonucleotide and oligonucleotide analogue drugs based on binding to specific nucleic acid sequences or proteins. In particular, oligonucleotide analog drugs are being researched and developed to improve resistance to nuclease, binding substances, binding ability and specificity.

Oligonucleotide analog drugs have been designed to improve resistance to nuclease, binding affinity with binding agents, and specificity. Among them, N3 '→ O5' phosphoramidates are stable in double helix and triple helix, and are known to be more resistant to nuclease than ordinary DNA or RNA (JK Chen et al, SM Gryaznov et al., Nucleic Acids Res., 24, 1508 (1996), Nucleic Acids Res., 23, 2661-2668 (1994), C. Escude et al., Proc Natl Acad Sci USA, 93, 4365-4369 -0514 (1996); C. Giovannangeli et al., Proc. Natl. Acad. Sci. USA, 94, 79-84 (1997)). They can be used as PCR primers with high selectivity for DNA hybridization probes and RNA sequences with low copy numbers. However, the biggest problem in using these compounds is that it is difficult to prepare 3'-amino-nucleoside.

In the chemical and enzymatic preparation of 3'-amino-2 ', 3'-amino-2', 3'-dideoxyguanosine (ADG) 3'-azido-3'-deoxythymidine (AZT) has been utilized. 3'-azido-3'-deoxy thymidine is produced from thymidine (TMD) as an important medicinal ingredient which has been proven to have anti-human immunodeficiency virus activity (N. Miller et al., J. Org. Chem. , 29, 1772-1776 (1964), Horwitz et al., J. Org. Chem., 29, 2076-2078 (1964)).

3'-amino-2 ', 3'-dideoxyguanosine is synthesized by a chemical process using 3'-azido-3'-deoxythymidine as a starting material and 3'-azido-3'-deoxy- -O- acetyl thymidine (3'-azido-3'-deoxy -5'-O-acetylthymidine) to N ^2, create-a method of replacement with palmitoyl guanine (N ² -palmitoylguanine) but known (M. Imazawa (JOC, 43 (15): 3044 (1978)), which is difficult to separate and purify due to the formation of an anomer.

Chemically and enzymatically, 3'-azido-3'-deoxythymidine is chemically reduced to form 3'-amino-2 ', 3'-deoxythymidine (3'-amino-2', 3 ' -deoxythymidine (ATMD), and Escherichia coli BMT-38 cells were treated with thymidine nucleoside phosphorylase and purine nucleoside phosphorylase (GV Zaitseva et al., Nucleosides & Nucleotides, 13 (1-3): 819 (1994)) are known to produce 3'-amino-2 ', 3'-dideoxyguanosine. However, in GV Zaitseva, 250 mg (1.04 mmol) of 3'-amino-3'-deoxy thymidine, 320 mg (1.13 mmol) of guanosine and 30 mg of dried E. coli E. coli BMT-38) was added and reacted at 50 ° C for 28 hours. Then, 56.7 mg (0.213 mmol) of 3'-amino-2 ', 3'-dideoxy guanosine And reported that the efficiency was very low despite the yield of 20.5%.

Also, U.S. Published Patent Application No. 2007/0065922 discloses a method for producing Escherichia coli (Escherichia coli) which shows high activity against thymidine phosphylase (TPase) and purine nucleoside phosphorylase (PNPase) 1K / 1T) was selected to perform the reaction. Amino-3'-deoxythymidine 100 mM (2.42 g) and 2,6-diaminopurine (DAP) 50 mM in 20 mM phosphate buffer (pH 6.0) (1250 IU) and PNPase (950 IU) were added to each well, and the mixture was incubated at 50 ° C. for 3 hours at 37 ° C., After reacting for 16 to 24 hours, purification was carried out using an ion exchange resin to obtain a solution of 3'-amino-2 '(3'-amino-3'-deoxythymidine) , 3'-dideoxy-2,6-diaminopurine riboside (ADDAP). Subsequently, 167 mM (2.65 g) of 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside prepared as described above, adenosine deaminase (5 mg / Boehringer Mannheim) was added and reacted at 50 ° C or less for 24 to 48 hours. After purification, 2.46 g of 3'-amino-2 ', 3'-dideoxy guanosine was prepared. That is, it is described that the initial 3'-amino-3'-deoxythymidine was produced in a molar yield of about 38% from the final 3'-amino-2 ', 3'-dideoxy guanosine.

However, the above-mentioned biosynthetic methods for biosynthesis have the following problems and their commercial use is very limited.

First, the molar yield of up to about 38% based on the main raw material, 3'-amino-3'-deoxythymidine, is a very inefficient method for industrial application.

Second, the added amount of microorganism cells used to convert 10 mmol (2.42 g) of 3'-amino-3'-deoxythymidine reaches about 3 g, and when such a large amount of microorganism is used, And industrialization is not possible due to the reduction of the process time and yield consumed to remove the cells after completion of the bioconversion.

Third, in the above-mentioned documents, the reaction was performed by setting the concentration of the main raw material to a low concentration of 10 mM to 100 mM. However, such a concentration is not industrially applicable. Accordingly, it is necessary to establish a high concentration of reaction conditions in order to improve facility efficiency and production efficiency. Prior art has not disclosed any of these reaction conditions at all.

Fourth, the enzymatic conversion method requires pretreatment of glutaraldehyde in order to prevent destruction of microbial cells at relatively high temperature conditions of 50 ° C. (PH Ninh et al., Appl. Environ. Microbiol., 79 (6), 1996 -2001 (2013)).

Fifth, in the above-mentioned documents, the molar ratio between the reaction substrates is 2 to 3 times in order to increase the sugar transfer yield, and after the enzyme conversion is completed, a large amount of unreacted material is contained, .

Sixth, a purification method using an ion exchange resin also requires a separate resin tower in the process design, and the prolongation of the process due to adsorption, desorption, and addition of a concentration process of the product fraction has a problem of increasing manufacturing cost of the product.

Under these circumstances, it is necessary to research and optimize the selection of appropriate enzymes and the sugar transfer reaction which can reduce the difference in molar ratio between substrates at high concentration and increase the sugar transfer yield. Furthermore, it is necessary to develop a method for purifying 3'-amino-2 ', 3'-dideoxyguanosine which is economically and efficiently pure in the reaction solution in which the bioconversion is completed as described above.

The present invention relates to (a) a bacteriophage derived polynucleotide phosphorylase derived from bacillus stearothermophilus and a bacteriostatic mothylated pyrimidine nucleoside phosphorylase derived from bacillus stearothiocyanate and a 3'-amino-3'-deoxythymidine Diaminopurine to produce 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside; And

(b) 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside was treated with adenosine deaminase derived from lactococcus lactis to form 3'-amino-2' Amino-2 ', 3'-dideoxyguanosine comprising the steps of: preparing 3'-amino-2'-dideoxy guanosine.

The present invention also relates to a process for preparing 3'-amino-2 ', 3'-dideoxy guanosine in step (b), followed by (c) adding alcohol and a strong base to the reaction product of step (b) Amino-2 ', 3 ' -dicarboxylic acid, which further comprises removing an enzyme source and a reaction by-product of a phosphorylase, a seed phosphorylase, a pyrimidine nucleoside phosphorylase and an adenosine deaminase. Dideoxy guanosine. ≪ / RTI >

As a result of intensive efforts to develop a production method for the industrial mass production of 3'-amino-2 ', 3'-dideoxyguanosine, the present inventors have found that Bacillus stearothermophilus-derived purinuclease seed phosphorylase and Bacillus stearate Amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside was produced in high yield by using a mophilus-derived pyrimidine nucleoside phosphorylase as an enzyme, and Lactococcus lactis Amino-2 ', 3'-dideoxy guanosine can be produced at a high yield in comparison with a known method when treating adenosine deaminase derived from horse chestnut, and completed the present invention.

The present invention relates to (a) a bacteriophage derived polynucleotide phosphorylase derived from bacillus stearothermophilus and a bacteriostatic mothylated pyrimidine nucleoside phosphorylase derived from bacillus stearothiocyanate and a 3'-amino-3'-deoxythymidine Diaminopurine to produce 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside; And

(b) 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside was treated with adenosine deaminase derived from lactococcus lactis to form 3'-amino-2' Amino-2 ', 3'-dideoxyguanosine comprising the steps of: preparing 3'-amino-2'-dideoxy guanosine.

In the present invention, 3'-amino-2 ', 3'-dideoxy guanosine is a compound having a structure represented by the following formula (1).

[Chemical Formula 1]

In the present invention, 3'-amino-3'-deoxythymidine is a compound having a structure represented by the following formula (2).

(2)

In the present invention, 2,6-diaminopurine is a compound having a structure represented by the following formula (3).

(3)

In the present invention, 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside is a compound having the structure represented by the following formula (4).

[Chemical Formula 4]

In the present invention, thymine is a compound having a structure represented by the following formula (5).

[Chemical Formula 5]

In the present invention, a nucleoside phosphorylase (NP) collectively refers to an enzyme that phosphorylates an N-glycosidic linkage of a nucleoside in the presence of phosphoric acid , The reaction represented by the following formula is catalyzed.

Ribonucleoside  + Phosphoric acid -> nucleic acid base + Ribos -1-phosphoric acid

The method for producing 3'-amino-2 ', 3'-dideoxy guanosine according to the present invention is a method for producing 3'-amino-2', 3'-dideoxyguanosine using Bacillus stearothermophilus- derived nucleoside phosphorylase. Oxy-2, 6-diaminopurine riboside, and thus provides an intermediate capable of efficiently producing 3'-amino-2 ', 3'-dideoxy guanosine. Particularly, when the enzyme source of the present invention is used, the production method using purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase derived from other sources, the production method using other nucleoside phosphorylase, Amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside.

The bacillus stearothermophilus-derived purinucleoside phosphorylase and the bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase according to the present invention can be synthesized by reacting 3'-amino-3'-deoxythymidine and 2, 6-diaminopurine is described in the following two-step reaction.

1) Pyrimidine nucleoside phosphorylase reaction

3'-amino-3'- Deoxy thymidine  + Phosphoric acid → thymine + 3-amino-2,3- Dideoxyribose -1-phosphoric acid

2) Purin  Nucleoside phosphorylase reaction

2,6- Diaminopurine  + 3-Amino-2,3- Dideoxyribose -1-phosphate → 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside + phosphoric acid

As in the above reaction, the pyrimidine nucleoside phosphorylase displaces 3'-amino-3'-deoxythymidine with 3-amino-2,3-dideoxyribose-1-phosphate, Amino-2,3-dideoxyribose-1-phosphate is converted to 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside by purine nucleoside phosphorylase .

In the present invention, adenosine deaminase is an enzyme that removes amino groups from the adenine portion of adenosine by hydrolysis to produce inosine, and catalyzes the reaction represented by the following formula.

3) adenosine Diaminase  The reaction (hydrolysis of the amino group)

3'-amino-2 ', 3'- Dideoxy -2,6- Diaminopurin riboside → 3 '-Amino-2', 3'-dideoxy guanosine

As in the above reaction, adenosine deaminase hydrolyzes the amino group of the adenine portion of 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside to form 3'- , 3'-dideoxy guanosine.

In the present invention, purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase are purine nucleoside phosphorylase derived from Bacillus stearothermophilus and pyrimidine nucleoside phosphorylase derived from Bacillus stearothermophilus . It is Ajay. According to one embodiment of the present invention, the Bacillus stearothermophilus is Bacillus stearothermophilus TH6-2 (FERM BP-2758).

In the present invention, adenosine deaminase is adenosine deaminase derived from Lactococcus lactis . According to one embodiment of the invention, the Lactococcus lactis is Lactococcus lactis KCCM 40104.

By analyzing the molecular biological properties and amino acid sequence of purine nucleoside phosphorylase derived from Bacillus stearothermophilus and / or pyrimidine nucleoside phosphorylase described above by molecular biology and gene engineering, A gene is obtained from Bacillus stearothermophilus to construct a recombinant plasmid in which a control region necessary for expression of the gene and the gene is inserted, and introduced into an arbitrary host to produce a recombinant strain in which the protein is expressed. Accordingly, the genetic recombinant strains in which a purine nucleoside phosphorylase derived from Bacillus stearothermophilus and / or a pyrimidine nucleoside phosphorylase gene are introduced into any host are included in the scope of the present invention.

Also, by analyzing the molecular biological characteristics and amino acid sequence of adenosine deaminase derived from lactococcus lactis, the gene of the protein is obtained from the lactococcus lactis and the recombination A plasmid can be constructed and introduced into an arbitrary host to produce a recombinant strain in which the protein is expressed. Accordingly, a gene recombinant strain in which an adenosine deaminase gene derived from lactococcus lactis is introduced into an arbitrary host is included in the scope of the present invention.

 The purine nucleoside phosphorylase derived from Bacillus stearothermophilus of the present invention comprises a purine nucleoside phosphorylase of SEQ ID NO: 1 or a fragment thereof exhibiting its activity, wherein the purine nucleoside phosphorylase derived from Bacillus stearothermophilus-derived pyrimidine nucleoside The seed phosphorylase comprises a pyrimidine nucleoside phosphorylase of SEQ ID NO: 2 or a fragment showing the activity thereof, and the adenosine deaminase derived from lactococcus lactis comprises an adenosine deaminase of SEQ ID NO: Activity. ≪ / RTI >

Bacillus stearothermophilus-derived purine nucleoside phosphorylase can be synthesized from the nucleotide sequence of SEQ ID NO: 4.

Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase can be synthesized from the nucleotide sequence of SEQ ID NO: 5.

Adenosine deaminase derived from lactococcus lactis can be synthesized from the nucleotide sequence of SEQ ID NO: 6.

The recombinant plasmid refers to a gene construct comprising an essential regulatory element operatively linked to the expression of the gene insert. The control region necessary for the expression includes a nucleic acid expression control sequence and a desired protein (including a promoter sequence (including an operator sequence for controlling transcription), a ribosome binding sequence (SD sequence), a transcription termination sequence, Is contained in the nucleic acid sequence.

Specific examples of the promoter sequence include trp promoter of tryptophan operon derived from Escherichia coli, lac promoter of lactose operon, promoter derived from lambda phage, or gluconic acid synthase promoter derived from Bacillus subtilis promoter gnt, an alkaline protease promoter apr and a neutral protease promoter npr α-amylase promoter amy. Sequences that have been independently modified or designed, such as the tac promoter, may also be used.

The ribosome binding sequence may be a sequence derived from Escherichia coli or Bacillus subtilis, but is not particularly limited as long as it functions in an intended host such as Escherichia coli or Bacillus subtilis. For example, a consensus sequence in which 4 or more bases of a sequence complementary to the 3 'terminal region of 16S ribosomal RNA is consecutively produced by DNA synthesis may be used.

The transcription termination sequence may be any one that is non-ρ factor dependent, for example, a lipoprotein terminator, a trp operon terminator, or the like.

Sequence order on the recombinant plasmids of these control regions is preferably arranged in the order of the promoter sequence, ribosome binding sequence, nucleoside phosphorylase or adenosine deaminase-encoding gene and transcription termination sequence from the 5 'terminal side Do.

Examples of the plasmid include, but are not limited to, pFRPT (Korean Patent No. 10-0449639), pBR322, pUC18, Bluescript II SK (+), pKK223-3, or pSC101 having autonomously replicable regions in E. coli, PVZ10, pTZ4, pC194, p11 or phi 1 - phi 105 having autonomous replicable regions in the host can be used. Examples of plasmids capable of autonomous replication in two or more host cells include pHV14, TRp7, YEp7 or pBS7 There is a number.

Said optional hosts include, but are not limited to, Escherichia coli), Bacillus subtilis (Bacillus subtilis) comprising a Bacillus sp, such as, preferably, there is industrially the E. coli (Escherichia coli) by using the easily be used. According to one embodiment of the present invention, the above host is Escherichia coli JM109.

In the present invention, the purine nucleoside phosphorylase derived from Bacillus stearothermophilus, the pyrimidine nucleoside phosphorylase derived from Bacillus stearothermophilus and / or the adenosine deaminase derived from lactococcus lactis Usable forms include enzyme itself, a cell having an enzyme activity, a cell treated product, or a fixed product thereof, and the reaction can be carried out using the enzyme. The microbial cells may be microbial cells separated by centrifugation or lyophilized microbial cells. The cell-processed product includes a cell lysate produced by, for example, acetone-dried cells, mechanical destruction, ultrasonic disruption, freeze-thaw treatment, pressure reduction treatment, osmotic pressure treatment, autolysis, cell wall decomposition treatment, , And further includes a microorganism treated by ammonium sulfate precipitation or acetone precipitation or column chromatography as necessary.

According to one embodiment of the present invention, the inventors of the present invention have found that the gene for purine nucleoside phosphorylase derived from Bacillus sp. And / or pyrimidine nucleoside phosphorylase for E. coli expression vector pFRPT (Korean Patent No. 0449639) And introduced into E. coli JM109 to complete pFRPT-BPUNP / JM109 and pFRPT-BPYNP / JM109 recombinant strains. In addition, the gene for adenosine deaminase derived from lactococcus lactis was inserted into the expression vector pFRPT for Escherichia coli (Korean Patent No. 0449639) and introduced into E. coli JM109 to complete a pFRPT-LADD / JM109 recombinant strain .

The dried bacterial cells recovered through the culturing process of the completed recombinant strain and centrifugal separation were recovered by freeze-drying treatment on the wet or microbial cells recovered. 3'-amino-2 ', 3'-dideoxy guanosine can be produced using the above-prepared wet cells.

(A) a bacteriophage-derived polynucleotide phosphorylase derived from bacillus stearothermophilus and a bacteriostea-derived pyrimidine nucleoside phosphorylase derived from bacillus stearothiocyanate, and 3'-amino-3'-deoxythymidine and Diaminopurine to produce 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside; And (b) treating the 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside with adenosine deaminase derived from lactococlu lactose to obtain 3'-amino- 3'-dideoxy guanosine can efficiently produce 3'-amino-2 ', 3'-dideoxy guanosine through the reaction under the following conditions.

(A) a bacteriophage-derived polynucleotide phosphorylase derived from bacillus stearothermophilus and a bacteriostea-derived pyrimidine nucleoside phosphorylase derived from bacillus stearothiocyanate, and 3'-amino-3'-deoxythymidine and In the step of preparing 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside by treatment with 2,6-diaminopurine, the reaction temperature is lower than 50 ° C, , Preferably about 40 < 0 > C. 3-amino-2,3-dideoxyribose-1-phosphate is reacted with amino-carbonyl (amino-carbonyl) carbonyl reaction). These results lead to a decrease in the yield of the final product, 3'-amino-2 ', 3'-dideoxy guanosine, and the impurities produced by browning were reduced during purification by 3'-amino-2', 3'- Which causes the crystallization of oxyguanosine to be disturbed. Therefore, it is preferable to conduct the reaction at a reaction temperature of about 40 캜.

(A) a bacteriophage-derived polynucleotide phosphorylase derived from bacillus stearothermophilus and a bacteriostea-derived pyrimidine nucleoside phosphorylase derived from bacillus stearothiocyanate, and 3'-amino-3'-deoxythymidine and In the step of preparing 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside by treatment with 2,6-diaminopurine, the reaction pH is pH 7.5 to 9.5, preferably pH 8.0 to 9.0, the closer the pH is to neutrality, the more stable the enzyme can be, preferably 8.0 to 8.5.

(A) a bacteriophage-derived polynucleotide phosphorylase derived from bacillus stearothermophilus and a bacteriostea-derived pyrimidine nucleoside phosphorylase derived from bacillus stearothiocyanate, and 3'-amino-3'-deoxythymidine and In the step of preparing 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside by treatment with 2,6-diaminopurine, the reaction time may vary depending on the enzyme throughput, 24 hours to 96 hours, preferably 30 hours to 48 hours.

(A) a bacteriophage-derived polynucleotide phosphorylase derived from bacillus stearothermophilus and a bacteriostea-derived pyrimidine nucleoside phosphorylase derived from bacillus stearothiocyanate, and 3'-amino-3'-deoxythymidine and In the step of preparing 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside by treatment with 2,6-diaminopurine, the concentration of the substrate is adjusted to 1 M or more, -Amino-3'-deoxythymidine and 2,6-diaminopurine. That is, the reaction proceeds smoothly in a high-concentration substrate and provides a manufacturing method suitable for industrial manufacturing process development.

It is preferable that 3'-amino-3'-deoxythymidine and 2,6-diaminopurine are contained in the substrate in a molar ratio of 1: 1. By having a molar ratio of 1: 1, it is possible to reduce the waste of raw materials and to facilitate the purification in the subsequent process. This reduces the waste of raw materials and facilitates purification in the subsequent process as compared to the known processes increasing the equivalence ratio of a particular substrate to perform the conversion reaction.

The bacillus stearothermophilus-derived purinucleoside phosphorylase and the bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase according to the present invention can be synthesized by reacting 3'-amino-3'-deoxythymidine and 2, The step of reacting with 6-diaminopurine may further be carried out in the presence of phosphoric acid or a salt thereof.

The 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside (b) of the present invention is treated with adenosine deaminase derived from lactocokus lactis, ', 3'-dideoxyguanosine, the reaction can be carried out directly following the step (a) reaction without a separate purification process. That is, a preparation of the nucleoside phosphorylase used for the conversion of the intermediate 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside, or a 3'- , And 3'-dideoxy-2,6-diaminopurine riboside are directly added to the reaction solution without any separate purification process, the reaction process is shortened and the overall reaction yield is greatly increased . Therefore, adenosine deaminase derived from lactococcus lactis can be directly administered without purification of the reactant in step (a).

The 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside (b) of the present invention is treated with adenosine deaminase derived from lactocokus lactis, ', 3'-dideoxyguanosine, the reaction pH may be 7.0 to 7.5. In order to maintain such a pH, the reaction may be carried out by adding a weak acid aqueous solution, for example, an aqueous acetic acid solution.

The 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside (b) of the present invention is treated with adenosine deaminase derived from lactocokus lactis, Dideoxy guanosine, the reaction temperature is 30 to 50 ° C, preferably 45 ° C or less, more preferably 40 ° C or less, still more preferably 30 to 40 ° C, most preferably about 40 Lt; 0 > C.

The 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside (b) of the present invention is treated with adenosine deaminase derived from lactocokus lactis, ', 3'-dideoxy guanosine, the reaction time may be from 24 hours to 120 hours.

The present invention also relates to a process for preparing 3'-amino-2 ', 3'-dideoxy guanosine in step (b), followed by (c) adding alcohol and a strong base to the reaction product of step (b) Amino-2 ', 3 ' -dicarboxylic acid, characterized in that it further comprises the step of removing the enzyme source and reaction by-products of the seed phosphorylase, the pyrimidine nucleoside phosphorylase and the adenosine deaminase. Dideoxy guanosine is provided.

The process for producing 3'-amino-2 ', 3'-dideoxy guanosine according to the present invention is a process for producing 3'-amino-2', 3'-dideoxy guanosine in high yield without purification by ion exchange resin, Exchange resins and the like, it is possible to solve the disadvantage that the process time is prolonged by adsorption, desorption, and concentration of liquids and is preferable for industrial mass production. That is, 3'-amino-2 ', 3'-dideoxy guanosine purified at a high purity can be produced at a high yield through the above steps.

In the method for producing 3'-amino-2 ', 3'-dideoxy guanosine according to the present invention, (c) an alcohol and a strong base are added to the reaction product of step (b) to form purine nucleoside phosphorylase, The step of removing the enzyme source and the reaction by-products of the amine nucleoside phosphorylase and the adenosine deaminase is carried out under the following conditions: purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase and adenosine deaminase Amino-2 ', 3'-dideoxy guanosine can be efficiently produced by simultaneously removing the enzyme source and the reaction by-products, especially thymine.

(c) adding an alcohol and a strong base to the reactant in step (b) to remove an enzyme source of the purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, and adenosine deaminase and reaction by-products Is a by-product of the reaction, which is an unreacted substrate and thymine and guanine, which are main byproducts produced through the reaction, and a purine nucleoside phosphorylase derived from bacillus stearothermophilus, Bacillus stearothermophilus originated pyrimidine nucleoside Phosphorylase and adenosine deaminase derived from lactococcus lactis are simultaneously removed.

(c) The step of adding alcohol and a strong base to the reaction product of step (b) to remove reaction by-products is generally carried out by filtration using an ultrafiltration membrane (UF), a membrane filter (MF) Filtration, continuous centrifugation, and diatomaceous filtration to filter the cells, and diatomaceous earth can be preferably used.

In order to perform the filtration, the substances other than the enzyme source are preferably dissolved in a solvent. Since a typical nucleoside has high solubility in an aqueous solution of a strong base, it is preferable to add a strong base after enzyme conversion to dissolve the nucleosides and bases excluding the cells, and then subject the cells to filtration. On the other hand, unlike nucleoside and threonine, thymine was found to be very low solubility in alcohol of strong bases, so that reaction by-products such as thymine can be removed together with an alcohol suspension of a strong base.

Preferably, the reactant in the step of preparing 3'-amino-2 ', 3'-dideoxyguanosine is treated with diol dissolved in strong base and diatomaceous earth to remove thymine from the reaction solution and the reaction by-product. Simultaneous removal of thymine and enzyme source is advantageous in that it simplifies the process and also reduces the time of the separation process by improving the filtration ability of thymine by collecting the cells in comparison with the process of removing only the cells.

The strong base may be any one or more selected from sodium hydroxide, potassium hydroxide, calcium hydroxide and barium hydroxide, and the alcohol is preferably a lower alcohol having 1 to 4 carbon atoms, preferably methyl alcohol, ethyl alcohol, 1-propyl alcohol and 2- Propyl alcohol. ≪ / RTI > Preferably, the alcohol suspension of the strong base is preferably 13 to 15 times methyl alcohol and 0.6 to 0.8 times caustic soda, based on the amount of 3'-amino-3'-deoxythymidine introduced.

(c) filtering the by-product in the step of removing by-products by adding an alcohol and a strong base to the reactant in the step (b), the filtrate may be neutralized (pH7.5 to 8.0) using hydrochloric acid, The crystallization is induced by stirring at an elevated temperature and slowly cooled, and the crystalline product is filtered to recover the primary crystallization product, thereby increasing the purity.

Purified water and basic charcoal (β-charcoal) can be further processed to increase the purity of the primary crystallization product. Amino-3'-deoxytrimidine was prepared by heating an aqueous solution of 8 to 12 N sodium hydroxide under purified water at a rate of 15 to 20 times the amount of 3'-amino-3'-deoxythymidine in the temperature range of 70 to 80 ° C. A basic charcoal of 0.05 to 0.3 times as much as the amount of the dean charged can be charged and stirred and filtered under high temperature conditions.

Through this purification process, the content of guanine in the byproducts is greatly reduced.

The above step can be repeatedly carried out to produce 3'-amino-2 ', 3'-dideoxy guanosine in high purity, and a 0.2 μm membrane (MF) filtration step is added between each crystallization step, And the coagulated solid of the water-soluble protein derived from the cells can be removed.

According to the method for producing 3'-amino-2 ', 3'-dideoxy guanosine according to the present invention, the nucleoside phosphorylase derived from bacillus stearothermophilus and the adenosine deaminase derived from lactocokus lactis are used Amino-2 ', 3'-dideoxyguanosine was produced at a high yield by high conversion of 3'-amino-2', 3'-dideoxyguanosine, 3'-amino-2 ', 3'-dideoxyguanosine is produced in high purity without purification, and thus 3'-amino-2', 3'-dideoxyguanosine can be mass-produced economically and efficiently.

1 shows a process for preparing 3'-amino-2 ', 3'-amino-2', 3'-dideoxyguanosine according to the present invention.
Figure 2 shows the structure of the pFRPT-BPUNP expression vector prepared in the present invention.
Figure 3 shows the structure of pFRPT-BPYNP expression vector prepared according to the present invention.
Figure 4 shows the structure of pFRPT-LADD expression vector prepared according to the present invention.
FIG. 5 shows the conversion rate over time according to the conversion of 3'-amino-2 ', 3'-dideoxy guanosine to the enzyme of the present invention.
Fig. 6 shows the results of HPLC analysis after conversion of 3'-amino-2 ', 3'-dideoxy guanosine to the enzyme of the present invention.
FIG. 7 shows the results of HPLC analysis of the substance recovered after conversion and final purification of 3'-amino-2 ', 3'-dideoxy guanosine of the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following embodiments are only examples for helping understanding of the invention, and thus the scope of the present invention is not limited thereto.

< Example  1> Bacillus Stearo mothyl  origin Purin  Purine nucleoside &lt; RTI ID = 0.0 &gt; phosphorylase ) &Lt; / RTI &gt;

Purine nucleoside phosphorylase derived from Bacillus (BPUNP), which is used for transglycosylation in the preparation of 3'-amino-2 ', 3'-dideoxyguanosine, Over-expressing transformed E. coli was prepared by the following method.

Bacillus stearothermophilus TH 6-2 ( Bacillus stearothermophilus Synthesis of a 705 bp oligonucleotide corresponding to the gene sequence of TH 6-2 (FERM BP-2758) (Genebank Accession Number D87960, nucleotides 619-1323, SEQ ID NO: 4) and cloning into pUC57 were performed by Macrogen , www.macrogen.com) to obtain pUC57-BPUNP. In addition, primers for PCR (SEQ ID NOS: 7 and 8) for cloning into an expression vector were designed as follows, and they were prepared and used by Bionics Inc. (Korea, www.bionicsro.co.kr).

[Table 1] BPUNP  synthesis primer  order

Using the above pUC57-BPUNP as a template, 200 μM of dNTP, 20 pmol of primer, 1 × Taq DNA polymerase buffer and 2.5 U of Taq DNA polymerase (TaKaRa Ex Taq, Cat. # RR001A, TAKARA, Japan, www. The reaction was repeated 30 times at 94 ° C for 30 seconds, at 50 ° C for 1 minute, and at 72 ° C for 1 minute under the condition of Takara-bio.com). As a result, the amplified 723 bp PCR product was confirmed by agarose gel electrophoresis, and this was extracted with a gel extraction kit (QIAquick Gel Extraction Kit, cat. # 28704, Qiagen, Germany, www.qiagen.com) . The purified DNA fragment was ligated to pGEM-T easy vector (pGEM-T easy vector system II, cat. # A1380, Promega, USA, www.promega.com), and ligated into JM109 E. coli cells (pGEM-T easy vector system II), colonies containing the desired plasmid were selected and named as pGEM-BPUNP / JM109. The pGEM-BPUNP / JM109 strain was inoculated in 3 ml of the medium shown in Table 2, cultured overnight at 37 DEG C, 200 rpm and pH 7, and centrifuged to harvest the cells. PGEM-BPUNP was isolated using plasmid purification kit (Dyne Plasmid Miniprep Kit, cat. # A510, Dainbio, Korea, www.dynebio.co.kr).

[Table 2] pGEM - BPUNP / JM109 strain culture medium component

10 μg of pFRPT was digested with 10 U of NdeI (cat. # 1161A, Cat. # 1161A) to insert the prepared Bacillus stearothermophilus-derived purinuclease seed phosphorylase into E. coli expression vector pFRPT (Korean Patent No. 0449639) TAKARA, Japan, www.takara-bio.com) and 10 U of XbaI (cat. # 1093A, TAKARA, Japan, www.takara-bio.com), and the excised plasmid was digested with agarose Gel electrophoresis, and 6.45 kbp fragments were purified using a gel extraction kit. On the other hand, 10 ug of pGEM-BPUNP was digested with 20 쨉 l of a reaction solution containing 10 U of NdeI and 10 U of XbaI and analyzed by agarose electrophoresis as described above. The 716 bp BPUNP DNA fragment was purified using a gel extraction kit Respectively.

Two DNA fragments obtained by the above method were added to a reaction solution containing 3 U of ligase (T4 DNA ligase, cat. # 2011A, TAKARA, Japan, www.takara-bio.com) and 1X ligase buffer to obtain 16 Lt; 0 &gt; C for 18 hours. The plasmid was extracted from E. coli colonies obtained by transforming JM109 E. coli cells with the above reaction solution, and the plasmid in which the desired DNA fragment was inserted was designated as pFRPT-BPUNP, and a schematic diagram thereof is shown in FIG. The transformant strain obtained by the above method was named pFRPT-BPUNP / JM109.

< Example  2> Bacillus Stearo mothyl  Derived Pyrimidine nucleoside &lt; RTI ID = 0.0 &gt; phosphorylase ) &Lt; / RTI &gt;

Pyrimidine nucleoside phosphorylase derived from Bacillus (BPYNP), which is used for transglycosylation in the production process of 3'-amino-2 ', 3'-dideoxyguanosine, Lt; / RTI &gt; was over-expressed by the following method.

A 1302 bp oligonucleotide corresponding to the gene sequence of Bacillus stearothermophilus TH 6-2 (FERM BP-2758) (Genebank Accession Number D87961, nucleotide number 138-1439, SEQ ID NO: 5) And cloning into pUC57 was carried out through a contract manufacturer, Macrogen, to obtain pUC57-BPYNP. In addition, a primer for PCR was designed (SEQ ID NOS: 9 and 10) for cloning into an expression vector, and was designed and used by Bionics as follows.

[Table 3] BPYNP  synthesis primer  order

Using the above pUC57-BPYNP as a template, PCR was carried out in the presence of 200 μM dNTP, 20 pmol of primer, 1 × Taq DNA polymerase buffer and 2.5 U of Taq DNA polymerase at 94 ° C. for 1 minute, Min, 72 째 C for 2 minutes for 30 times to perform PCR reaction. As a result, the amplified 1.3 kbp PCR product was confirmed by agarose gel electrophoresis and purified using a gel extraction kit. The purified DNA fragment was ligated to a pGEM-T easy vector, and a clone containing the desired plasmid was selected from Escherichia coli colonies obtained by transforming JM109 E. coli cells, and designated as pGEM-BPYNP / JM109. The pGEM-BPYNP / JM109 strain was inoculated with 3 ml of the same components as the medium of Table 2, cultured overnight at 37 ° C, 200 rpm, pH 7, and centrifuged to harvest the cells. The harvested microorganism was isolated from pGEM-BPYNP using a plasmid purification kit.

To insert the prepared Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase into E. coli expression vector pFRPT, 10 μg of pFRPT was digested with 10 U of NdeI and 10 U of HindIII (cat. # 1060A, TAKARA , Japan, www.takara-bio.com), and the digested plasmid was analyzed by agarose gel electrophoresis and the 6.45 kbp fragment was purified using a gel extraction kit. On the other hand, 10 ug of pGEM-BPYNP was digested with 20 ul of a reaction solution containing 10 U of NdeI and 10 U of HindIII, and analyzed by agarose electrophoresis as described above. The 1.3 kbp BPYNP DNA fragment was extracted with a gel extraction kit .

The two DNA fragments obtained by the above method were added to a reaction solution containing 3 U of ligase and 1X ligase buffer, and reacted at 16 DEG C for 18 hours. The plasmid was extracted from E. coli colonies obtained by transforming JM109 E. coli cells with the above reaction solution, and the plasmid in which the desired DNA fragment was inserted was designated as pFRPT-BPYNP, and a schematic diagram thereof is shown in FIG. The transformant strain obtained by the above method was named pFRPT-BPYNP / JM109.

< Example  3> Lactococcus Lactis  Derived adenosine Diaminase  (adenosine deaminase) expression strain

The transformed E. coli overexpressing adenosine deaminase (LADD) derived from lactococcus lactis to be used for the deamidation reaction in the 3'-amino-2 ', 3'-dideoxyguanosine production process was &Lt; / RTI &gt;

Lactococcus the lactis) strains (used under pre-sale from the Korea Association of seed, KCCM40104) inoculated into 50 ml TSB (BactoTM Tryptic Soy Broth , cat. # 211825, BD, USA, www.bd.com) medium and cultured overnight at 30 ℃ The cells were harvested by centrifugation. Genomic DNA was isolated from the collected strains using a genome purification kit (Cat. # A1120, Promega, USA, www.promega.com) and used as a PCR template.

The primer for PCR was an oligonucleotide for synthesizing a sequence corresponding to the nucleotide sequence of the adenosine deaminase gene derived from lactococcus lactis (nucleotide numbers 287447 to 288505, SEQ ID NO: 6 (1059 bp) of Genebank Accession Number NC_002662) Nos. 11 and 12) were designed as follows and manufactured through a consignment manufacturer, Bionics.

[Table 4] LADD  synthesis primer  order

Using the above-mentioned lactococus genomic DNA as a template, PCR was carried out at 94 ° C for 1 minute, 50 μl of dNTP, 30 pmol of primer, 1 × Taq DNA polymerase buffer and 2.5 U of Taq DNA polymerase in the presence of 200 μM of dNTP, 1 min at 72 ° C for 1 min and 30 sec. As a result, amplified 1078 bp PCR product was confirmed by agarose gel electrophoresis and purified using a gel extraction kit. The DNA fragment purified using the pGEM-T easy vector was ligated to the pGEM-T easy vector, and a clone containing the desired plasmid was selected from Escherichia coli colonies obtained by transforming JM109 E. coli cells. The resulting plasmid was cloned into pGEM-LADD / JM109 Respectively. The pGEM-LADD / JM109 strain was inoculated in 3 ml of the medium of Table 1, cultured overnight at 37 ° C, 200 rpm, pH 7, and centrifuged to harvest the cells. The harvested microorganism was isolated from pGEM-LADD using a plasmid purification kit.

In addition, pGEM-LADD was digested with BamHI (cat. # 1010A, TAKARA, Japan, www.takara-bio.com) and HindIII for the overexpression of the enzyme protein and ligated with the plasmid expression vector pQE31 (Qiagen, Germany, www.qiagen.com) and transformed into JM109 E. coli cells, and the plasmid in which the desired DNA fragment was inserted was named pQE31-LADD.

Then, an additional PCR primer based on the pQE31-LADD nucleotide sequence and an oligonucleotide of SEQ ID NO: 13 were inserted into the expression vector pFRPT for Escherichia coli in order to insert the adenosine deaminase gene derived from the lactococcus lactis, And used in conjunction with SEQ ID NO: 12 above.

Using the above-described pQE31-LADD plasmid as a template, PCR was carried out at 94 ° C for 1 minute, 50 ° C for 30 minutes, in the presence of 200 μM dNTP, 30 pmol of primer, 1 × Taq DNA polymerase buffer and 2.5 U of Taq DNA polymerase For 1 minute and 72 &lt; 0 &gt; C for 1 minute and 30 seconds were repeated 30 times for PCR reaction. As a result, amplified 1093 bp PCR product was confirmed by agarose gel electrophoresis and purified using a gel extraction kit. Then, 10 μg of pFRPT was digested in 20 μl of a reaction solution containing 10 U of BglII (cat. # 1021A, TAKARA, Japan, www.takara-bio.com) and 10 U of HindIII, and the digested plasmid was ligated with an agarose gel Electrophoresis, and 6.45 kbp fragments were purified using a gel extraction kit.

On the other hand, 10 μg of the PCR product obtained using the above SEQ ID NO: 13 and SEQ ID NO: 12 was digested with 20 μl of a reaction solution containing 10 U of BglII and 10 U of HindIII, and subjected to agarose electrophoresis And 1093 bp of the adenosine deaminase DNA fragment derived from lactococcus lactis was purified using a gel extraction kit. Two DNA fragments obtained by this method were added to a reaction solution containing 3 U of ligase and 1X ligase buffer, and reacted at 16 ° C for 18 hours. The plasmid was extracted from E. coli colonies obtained by transforming JM109 E. coli cells with the above reaction solution, and the plasmid in which the desired DNA fragment was inserted was designated as pFRPT-LADD, and a schematic diagram thereof is shown in FIG. The transformant strain obtained by the above method was named pFRPT-LADD / JM109.

< Example  4> pFRPT - BPUNP / JM109 Wet cell  Produce

For the production of the pFRPT-BPUNP / JM109 wet cells, the strain was cultured according to the medium composition shown in Table 5 below.

[Table 5] pFRPT - BPUNP / JM109 &lt; / RTI &gt;

PFRPT-BPUNP / JM109 was inoculated into a 250 ml Erlenmeyer flask containing 25 ml of the medium of Table 5 and cultured overnight at 37 ° C and 240 rpm. 2 ml of the gastric culture was inoculated aseptically into 200 ml of the medium of Table 5, which was contained in a 1 L Ellenmeyer flask, and cultured with shaking at 37 ° C and 240 rpm. When the absorbance reached 0.8, IPTG (isopropyl-1-thio-β-D-galactopyranoside, manufactured by Carbosynth) was added to a concentration of 1 mM. The culture was further incubated for 3 hours with shaking. The resulting culture was centrifuged at 8000 rpm for 10 minutes and washed with 20 ml of 10 mM phosphate buffer. Thus, an enzyme source of purine nucleoside phosphorylase was obtained.

The unit activity of purin nucleoside phosphorylase from bacillus stearothermophilus was calculated as follows.

1) Reaction formula

Guanosine + phosphoric acid → guanine + ribose-1-phosphate

2) Calculation of activity

U = molar number of guanine (μmol) / reaction time (min)

< Example  5> pFRPT - BPYNP / JM109 Wet cell  Produce

The strains were cultured according to the medium composition of Table 5 for the preparation of pFRPT-BPYNP / JM109 wet cells.

PFRPT-BPYNP / JM109 was inoculated into a 250 ml Erlenmeyer flask containing 25 ml of the medium shown in Table 5 and cultured overnight at 37 ° C and 240 rpm. 2 ml of the gastric culture was inoculated aseptically into 200 ml of the medium of Table 5, which was contained in a 1 L Ellenmeyer flask, and cultured with shaking at 37 ° C and 240 rpm. When the absorbance reached 0.8, IPTG (isopropyl-1-thio-β-D-galactopyranoside, manufactured by Carbosynth) was added to a concentration of 1 mM. The culture was further incubated for 3 hours with shaking. The resulting culture was centrifuged at 8000 rpm for 10 minutes and washed with 20 ml of 10 mM phosphate buffer. Thus, an enzyme source of pyrimidine nucleoside phosphorylase was obtained.

Unit activity of pyrimidine nucleoside phosphorylase derived from bacillus stearothermophilus was calculated as follows.

1) Reaction formula

5-methyluridine + phosphoric acid → thymine + ribose-1-phosphate

2) Calculation of activity

U = number of moles of thymine (μmol) / reaction time (min)

< Example  6> pFRPT - LADD / JM109 Wet cell  Produce

The strains were cultured according to the medium composition of Table 5 for the preparation of pFRPT-LADD / JM109 wet cells.

2 ml of the culture obtained by inoculating pFRPT-LADD / JM109 into a 250 ml Erlenmeyer flask containing 25 ml of the medium of Table 5 and incubating at 37 ° C overnight at 240 rpm was added to a 1 L Erlenmeyer flask containing 200 ml of the medium of Table 5 Inoculated aseptically and cultured at 37 ° C with shaking at 240 rpm. When the absorbance reached 0.8, IPTG was added to a concentration of 1 mM. The culture was further incubated for 3 hours with shaking. The resulting culture was centrifuged at 8000 rpm for 10 minutes and washed with 20 ml of 10 mM phosphate buffer to use as an enzyme source of adenosine deaminase.

The unit activity of adenosine deaminase was calculated as follows.

1) Reaction formula

2,6-diaminopurine 2'-deoxyriboside → 2'-deoxyguanine (2'-deoxyguanine)

2) Calculation of activity

U = 2'-deoxyguanine molar number (μmol) / reaction time (min)

< Example  7 > The reaction temperature of 3'-amino-2 ', 3'- Dideoxy -2,6- Diamino purine Enzyme conversion confirmation of boside

3'-amino-3'-deoxythymidine (ATMD) was synthesized as a precursor for the synthesis of 3'-amino-2 ', 3'-dideoxyguanosine. 3'-amino-3'-deoxy thymidine was prepared by dissolving 1 Kg of 3'-azido-3'-deoxythymidine (AZT, 3.74 mol) in 7.8 L of acetonitrile and stirred with acetonitrile. 1.17 Kg of triphenylphosphine (4.45 mol) was added thereto. The mixture was stirred at room temperature for 4 hours, and then 1 L of distilled water was added. The mixture was stirred at room temperature for 4 hours, concentrated under reduced pressure, and 2 L of methanol was added thereto. This was stirred at room temperature for 8 hours and then filtered to collect crystals, which were dried to obtain 0.79 Kg of 3'-amino-3'-deoxythymidine (3.27 mol, molar yield to AZT of 87.4%).

17.42 g of 2,6-diaminopurine (DAP, 2.32 M), 20 g of the synthesized 3'-amino-3'-deoxythymidine (ATMD, 1.6 M), 50 ml of purified water and 0.8 g of phosphoric acid (600 U) pFRPT-BPUNP / JM109 wet cells and 6.3 g (4400 U) pFRPT-BPYNP / JM109 wet cells were added to the substrate solution containing sodium phosphate monobasic (0.134 M) The temperature was changed to 50 캜 and 60 캜, and the mixture was shaken for 72 hours. After the reaction, a high-performance liquid chromatography column: Inertsil ODS-3 (5 μm, diameter: 4.6 mm, length: 150 mm, manufactured by GL science); 10 mM sodium phosphate buffer (pH 8.0) containing 4% methanol, detection; And was measured using ultraviolet 254 nm absorption.

The conversion to 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside (ADDAP) was calculated as follows.

ADDAP  Conversion rate % ) = ( ADDAP HPLC area%  X 100) / ( ADDAP HPLC area%  + DAP  HPLC area% )

The conversion to 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside (ADDAP) is shown in Table 6 below.

[Table 6] 3'-amino-2 ', 3'- Dideoxy -2,6- Diaminopurin riboside  Conversion Rate

As can be seen in Table 6, the highest conversion was observed at 40 ° C., and the color of the reaction solution changed to brown or brown over time at reaction conditions of 50 ° C. or higher, and 3 'to 6-diaminopurine- -Amino-3'-deoxythymidine was decomposed in a considerable amount. Therefore, a temperature condition of about 40 캜 is set as a preferable reaction condition.

< Example  8 > 3'-amino-3'- Deoxythymidine and  2,6- Of diaminopurine  The molar ratio of 3'-amino-2 ', 3'- Dideoxy -2,6- Of the diaminopurine riboside  Enzyme conversion confirmation

(58.0 mmol, 1.4 eq.), 7.49 g (49.9 mmol, 1.2 eq.) Of 2,6-diaminopurine feed based on 10 g (41.5 mmol) 3'- , And 6.25 g (41.6 mmol, 1 eq.). The suspension was suspended in 25 ml of 10 mM sodium dihydrogenphosphate solution to obtain 0.86 g (300 U) pFRPT-BPUNP / JM109 wet cells, 3.14 g pFRPT-BPYNP / JM109 wet cells were mixed and agitated for 5 days at 40 ° C. The conversion of 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside produced in the reaction solution was analyzed by HPLC in the same manner as in Example 7. The results are shown in Table 7 below .

[Table 7] Synthesis of 3'-amino-2 ', 3'- Dideoxy -2,6- Diaminopurin riboside  Conversion Rate

Amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside using a nucleoside phosphorylase derived from Bacillus stearothermophilus, the 3'-amino- The lower the equivalence of 2,6-diaminopurine based on 3'-deoxythymidine, the greater the conversion rate. The absolute value of the 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside produced was also high.

That is, the molar ratio of 3'-amino-3'-deoxythymidine to 2,6-diaminopurine was set to 1: 1 to minimize the formation of by-products after the reaction.

< Example  9 >, 3'-amino-2 ', 3'- Dideoxy -2,6- Diaminopurin riboside Enzyme conversion confirmation

12.5 g of 2,6-diaminopurine (83.2 mmol), 20 g of 3'-amino-3'-deoxythymidine (82.9 mmol), 50 ml of purified water and 0.06 g of sodium dihydrogenphosphate The pH of the reaction solution was adjusted to 7.6, 8.0, and 8.8 after mixing 1.7 g (600 U) pFRPT-BPUNP / JM109 wet cells and 6.3 g (4400 U) pFRPT-BPYNP / C &lt; / RTI &gt; for 24 hours. The conversion of 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside produced in the reaction solution was analyzed by HPLC in the same manner as in Example 7. The results are shown in Table 8 .

[Table 8] 3'-Amino-2 ', 3'- Dideoxy -2,6- Diaminopurin riboside  Conversion Rate

When the enzyme is converted to 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside by using a nucleoside phosphorylase derived from Bacillus stearothermophilus, the higher the pH, Of the total population. As a result, it was confirmed that the pH of the reaction solution was preferably between 8.0 and 9.0, and the closer to neutrality the more stable the enzyme was, and the preferable range was 8.0 to 8.5.

< Example  Amino-2 ', 3'- Dideoxy -2,6- Diaminopurine Enzyme conversion confirmation of riboside

To 82.5 mmol of 2,6-diaminopurine (83.2 mmol), 20 g of 3'-amino-3'-deoxythymidine (82.9 mmol) and 0.06 g of sodium hydrogen dihydrogenphosphate M substrate concentration) and 85 ml (1.0 M substrate concentration) purified water, respectively. The pH of the reaction mixture was adjusted to 8.0, and the mixture was shaken for 24 hours at 40 ° C. The pFRPT-BPYNP / JM109 cells were mixed with 1.7 g (600 U) pFRPT-BPUNP / JM109 wet cells and 6.3 g (4400 U) . The conversion of 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside produced in the reaction solution was analyzed by HPLC in the same manner as in Example 7. The results are shown in Table 9 .

[Table 9] Synthesis of 3'-amino-2 ', 3'- Dideoxy -2,6- Diaminopurin riboside  Conversion Rate

When the enzyme is converted to 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside using a nucleoside phosphorylase derived from bacillus stearothermophilus, the higher the substrate concentration And the conversion rate is increased. Further, the conversion reaction to 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside was carried out using a nucleoside phosphorylase derived from bacillus stearothermophilus at a concentration of 1.0 M or more It is confirmed that the process is excellent.

< Example  11> Lactococcus Lactis  Derived adenosine Diaminase  The 3'-amino-2 ', 3'- Dideoxy guanosine (ADG)  Enzyme conversion confirmation

12.5 g of 2,6-diaminopurine (83.2 mmol), 20 g of 3'-amino-3'-deoxythymidine (82.9 mmol), 50 ml of purified water and 0.06 g of sodium dihydrogenphosphate (600 U) pFRPT-BPUNP / JM109 wet cells and 6.3 g (4400 U) of pFRPT-BPYNP / JM109 wet cells were mixed, and the pH was adjusted to 8.0, followed by shaking stirring at 40 DEG C for 48 hours . Analysis of the reaction mixture by HPLC showed that the conversion of 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside was 70%. After conversion of 70% was confirmed by 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside, 20 ml of purified water was added and 0.4 g (1200 U) of pFRPT-LADD / JM109 The wet cells were mixed and agitated for 90 hours while maintaining the pH at 7.0 to 7.5 using a 50% aqueous solution of acetic acid at 40 ° C. The reaction rate was 99.65%.

The reaction rate was calculated as follows.

3'-amino-2 ', 3'- Dideoxy guanosine Reaction rate ( % ) = [( ADG HPLC area%  + ADDAP  HPLC area%) X 100] / (ADG HPLC area%  + ADDAP HPLC area%  + DAP HPLC area% )

Enzyme conversion through 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside by primary transfer using a nucleoside phosphorylase derived from bacillus stearothermophilus , And adenosine deaminase derived from lactococcus lactis was directly added to the reaction solution in which the reaction was carried out. Thus, it was confirmed that secondary enzyme conversion was possible with 3'-amino-2 ', 3'-dideoxy guanosine. Namely, it is possible to remove the nucleoside phosphorylase derived from bacillus stearothermophilus, or to purify the lacto-cocosyl phosphate without purification of the intermediate 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside It was confirmed that 3'-amino-2 ', 3'-dideoxy guanosine can be produced through a de-amination reaction in the same reaction solution using adenosine deaminase derived from lactis.

< Example  12> Cell and thymine removal

The reaction solution of Example 11 was divided into quarters and the amounts of caustic soda and methyl alcohol were varied to effect simultaneous removal of the cells and thymine (TMN).

A mixture of 0.9-fold caustic soda and 20-fold methyl alcohol (4.5 g caustic soda and 100 ml methyl alcohol), 0.65-fold caustic soda and 20-fold methyl alcohol (3.25 g caustic Soda and 100 ml methyl alcohol), 0.65-fold caustic soda and 14-fold methyl alcohol (3.25 g caustic soda and 70 ml methyl alcohol), 0.65-fold caustic soda and 10-fold methyl alcohol (3.25 g caustic soda and 50 ml methyl alcohol) After stirring at 40 ° C for 1 hour and slowly cooling, the mixture was kept at 4 ° C for 3 hours. After cooling and stirring, the cells and thymine were removed by filtration under reduced pressure.

The results of HPLC Area% according to the removal of the cells and thymine are shown in Table 10 below.

[Table 10] Thymine removal and 3'-amino-2 ', 3'- Dideoxy guanosine  Confirm

It was confirmed that thymine was removed except for the reaction solution using 0.65 times of caustic soda and 10 times of methyl alcohol (3.25 g of caustic soda and 50 ml of methyl alcohol) based on the added 3'-amino-3'-deoxy thymidine Respectively. In particular, the amount of thymine is largely removed by filtration, and the best condition for the content of 3'-amino-2 ', 3'-dideoxy guanosine in the filtrate is 3'-amino-2', 3'-dideoxy guanosine It was confirmed that the ratio of 0.65 times of caustic soda to 14 times of methyl alcohol.

< Example  13 > 3'-Amino-2 ', 3'- Dideoxy guanosine  Enzyme conversion confirmation

(207.39 mmol), 50 g of 3'-amino-3'-deoxythymidine (207.30 mmol), 125 ml of purified water and 0.15 g of sodium dihydrogen phosphate (1.25 mmol) After mixing 4.3 g (1500 U) of pFRPT-BPUNP / JM109 wet cells and 15.7 g (11000 U) of pFRPT-BPYNP / JM109 wet cells, pH was adjusted to 8.0, and the mixture was shaken at 40 ° C for 48 hours Lt; / RTI &gt; Analysis of the reaction mixture by HPLC showed that the conversion of 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside was 72.0%. After conversion of 70% or more was confirmed by 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside, 50 ml of purified water was added as a subsequent reaction, and 1 g (3000 U) of pFRPT- LADD / JM109 wet cells were mixed and agitated for 72 hours while maintaining the pH at 7.0 to 7.5 using a 50% aqueous acetic acid solution at 40 ° C. The reaction rate was 98.5%. The conversion of 3'-amino-2 ', 3'-dideoxyguanosine with time is shown in FIG. As shown in Fig. 5, it was confirmed that the reaction rate increased with time.

The results of HPLC analysis thereof are shown in Fig. According to the results of HPLC analysis, it was confirmed that 3'-amino-2 ', 3'-dideoxyguanosine and some by-products were detected together.

< Example  14 > in the reaction solution, 3'-amino-2 ', 3'- Dideoxy guanosine  Separation purification

700 ml of methyl alcohol dissolved in 32.5 g of caustic soda was added to the reaction solution of Example 13, and the mixture was stirred at 40 ° C for 1 hour and then cooled for 3 hours at 4 ° C. After cooling and stirring, the mixture was filtered through diatomaceous earth, Thymine was removed. The filtrate from which the cells and thymine were removed was adjusted to pH 8.1 with hydrochloric acid, stirred at 75 ° C for 1 hour, cooled at 35 ° C for 3 hours, and filtered. 800 ml of purified water was added to the thus obtained primary crystallization product, and the pH was adjusted to 10 with 10 N sodium hydroxide aqueous solution, and the temperature was raised to 75 캜 and stirred for 1 hour. After stirring at 75 ° C for 1 hour, 5 g of basic charcoal (β-charcoal) was added, and further stirred at 75 ° C for 1 hour and then hot-filtered at a high temperature.

In addition, the membrane filtration process of 0.2 μm was carried out simultaneously while the filtration was proceeding. The filtrate was adjusted to pH 7.5 with 50% aqueous hydrochloric acid solution and concentrated. The concentrated residue was suspended in 800 ml of ethyl alcohol, stirred at 75 ° C for 2 hours, slowly cooled, maintained at 35 ° C for 3 hours, stirred and filtered, and the filtrate was vacuum dried.

HPLC analysis was performed after the separation and purification, and the results are shown in FIG.

As shown in Figure 7, 37.9 g (142.3 mmol) of 3'-amino-2 ', 3'-dideoxyguanosine having a 99.1% HPLC purity was finally recovered, Corresponding to a weight yield of 75.8% based on the amount of deoxythymidine and a molar yield of 68.6%.

From the above results, it was confirmed that the method for producing 3'-amino-2 ', 3'-dideoxy guanosine according to the present invention is a suitable method for commercial mass production.

&Lt; 110 > ST Pharm Co., Ltd. <120> Methods for Preparing 3'-amino-2 ', 3'-dideoxyguanosine by Using          Nucleoside Phosphorylases Derived from Bacillus and Adenosine          Deaminase Derived from Lactococcus <130> P15-185-STP <160> 13 <170> KoPatentin 3.0 <210> 1 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> Purine nucleoside phosphorylase derived from Bacillus          stearothermophilus <400> 1 Met Ser Val His Ile Gly Ala Lys Glu His Glu Ile Ala Asp Lys Ile   1 5 10 15 Leu Leu Pro Gly Asp Pro Leu Arg Ala Lys Tyr Ile Ala Glu Thr Phe              20 25 30 Leu Glu Gly Ala Thr Cys Tyr Asn Gln Val Arg Gly Met Leu Gly Phe          35 40 45 Thr Gly Thr Tyr Lys Gly His Arg Ile Ser Val Gln Gly Thr Gly Met      50 55 60 Gly Val Pro Ser Ile Ser Ile Tyr Ile Thr Glu Leu Met Gln Ser Tyr  65 70 75 80 Asn Val Gln Thr Leu Ile Arg Val Gly Thr Cys Gly Ala Ile Gln Lys                  85 90 95 Asp Val Lys Val Arg Asp Val Ile Leu Ala Met Thr Ser Ser Thr Asp             100 105 110 Ser Gln Met Asn Arg Met Thr Phe Gly Gly Ile Asp Tyr Ala Pro Thr         115 120 125 Ala Asn Phe Asp Leu Leu Lys Thr Ala Tyr Glu Ile Gly Lys Glu Lys     130 135 140 Gly Leu Gln Leu Lys Val Gly Ser Val Phe Thr Ala Asp Met Phe Tyr 145 150 155 160 Asn Glu Asn Ala Gln Phe Glu Lys Leu Ala Arg Tyr Gly Val Leu Ala                 165 170 175 Val Glu Met Glu Thr Thr Ala Leu Tyr Thr Leu Ala Ala Lys Phe Gly             180 185 190 Arg Lys Ala Leu Ser Val Leu Thr Val Ser Asp His Ile Leu Thr Gly         195 200 205 Glu Glu Thr Thr Ala Glu Glu Arg Gln Thr Thr Phe Asn Glu Met Ile     210 215 220 Glu Val Ala Leu Glu Thr Ala Ile Arg Gln 225 230 <210> 2 <211> 433 <212> PRT <213> Artificial Sequence <220> <223> Pyrimidine nucleoside phosphorylase derived from Bacillus          stearothermophilus <400> 2 Met Arg Met Val Asp Leu Ile Glu Lys Lys Arg Asp Gly His Ala Leu   1 5 10 15 Thr Lys Glu Glu Ile Gln Phe Ile Ile Glu Gly Tyr Thr Lys Gly Asp              20 25 30 Ile Pro Asp Tyr Gln Met Ser Ala Leu Ala Met Ala Ile Phe Phe Arg          35 40 45 Gly Met Asn Glu Glu Glu Thr Ala Glu Leu Thr Met Ala Met Val His      50 55 60 Ser Gly Asp Thr Ile Asp Leu Ser Arg Ile Glu Gly Ile Lys Val Asp  65 70 75 80 Lys His Ser Thr Gly Gly Val Gly Asp Thr Thr Thr Leu Val Leu Gly                  85 90 95 Pro Leu Val Ala Ser Val Gly Val Pro Val Ala Lys Met Ser Gly Arg             100 105 110 Gly Leu Gly His Thr Gly Gly Thr Ile Asp Lys Leu Glu Ser Val Pro         115 120 125 Gly Phe His Val Glu Ile Thr Asn Asp Glu Phe Ile Asp Leu Val Asn     130 135 140 Lys Asn Lys Ile Ala Val Val Gly Gln Ser Gly Asn Leu Thr Pro Ala 145 150 155 160 Asp Lys Lys Leu Tyr Ala Leu Arg Asp Val Thr Ala Thr Val Asn Ser                 165 170 175 Ile Pro Leu Ile Ala Ser Ser Ile Met Ser Lys Lys Ile Ala Ala Gly             180 185 190 Ala Asp Ala Ile Val Leu Asp Val Lys Thr Gly Val Gly Ala Phe Met         195 200 205 Lys Asp Leu Asn Asp Ala Lys Ala Leu Ala Lys Ala Met Val Asp Ile     210 215 220 Gly Asn Arg Val Gly Arg Lys Thr Met Ala Ile Ile Ser Asp Met Ser 225 230 235 240 Gln Pro Leu Gly Tyr Ala Ile Gly Asn Ala Leu Glu Val Lys Glu Ala                 245 250 255 Ile Asp Thr Leu Lys Gly Glu Gly Pro Glu Asp Phe Gln Glu Leu Cys             260 265 270 Leu Val Leu Gly Ser His Met Val Tyr Leu Ala Glu Lys Ala Ser Ser         275 280 285 Leu Glu Glu Ala Arg His Met Leu Glu Lys Ala Met Lys Asp Gly Ser     290 295 300 Ala Leu Gln Thr Phe Lys Thr Phe Leu Ala Ala Gln Gly Gly Asp Ala 305 310 315 320 Ser Val Val Asp Asp Pro Ser Lys Leu Pro Gln Ala Lys Tyr Ile Ile                 325 330 335 Glu Leu Glu Ala Lys Glu Asp Gly Tyr Val Ser Glu Ile Val Ala Asp             340 345 350 Ala Val Gly Thr Ala Ala Met Trp Leu Gly Ala Gly Arg Ala Thr Lys         355 360 365 Glu Ser Thr Ile Asp Leu Ala Val Gly Leu Val Leu Arg Lys Lys Val     370 375 380 Gly Asp Ala Val Lys Lys Gly Glu Ser Leu Val Thr Ile Tyr Ser Asn 385 390 395 400 Arg Glu Gln Val Asp Asp Val Lys Gln Lys Leu Tyr Glu Asn Ile Arg                 405 410 415 Ile Ser Ala Thr Pro Val Gln Ala Pro Thr Leu Ile Tyr Asp Lys Ile             420 425 430 Ser     <210> 3 <211> 352 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > Adenosine deaminase derived from Lactococcus lactis <400> 3 Met Lys Lys Lys Arg Glu Lys Leu Val Leu Lys Ser Glu Ile Ile Ala   1 5 10 15 Gln Met Pro Lys Val Glu Leu His Cys His Leu Asp Gly Ser Leu Ser              20 25 30 Leu Ser Val Ile Lys Glu Leu Ala Lys Asn Ala Gly Ile His Met Thr          35 40 45 Met Ser Asp Glu Glu Ile Leu Glu Lys Ala Gln Ala Pro Glu Asn Thr      50 55 60 Lys Asn Leu Leu Glu Tyr Leu Gln Arg Phe Asp Phe Val Leu Pro Leu  65 70 75 80 Leu Gln Thr Tyr Lys Asn Leu Glu Leu Ala Ala Tyr Asp Val Val Arg                  85 90 95 Gln Ala Ala Asn Asp Asn Ile Lys Tyr Ile Glu Ile Arg Phe Ala Pro             100 105 110 Ser Gln His Leu Leu Glu Asn Leu Thr Leu Glu Glu Ala Val Glu Ala         115 120 125 Val Ile Ala Gly Leu Ser Arg Ala Glu Asn Asp Phe Asp Ile Arg Ala     130 135 140 Asn Ala Leu Val Cys Gly Leu Lys Gln Glu Pro Ile Gln Lys Leu Gln 145 150 155 160 Lys Leu Leu Pro Leu Phe Asp Lys Ile Pro Asp Glu His Leu Val Gly                 165 170 175 Phe Asp Met Ala Gly Asp Glu Leu Asn Tyr Pro Gln Glu Lys Phe Val             180 185 190 Asp Leu Ile His Asp Ile Lys Ile Lys Gly Val Asn Val Thr Leu His         195 200 205 Ala Gly Glu Cys Pro Ala Cys Glu Lys Asn Ile Leu Asp Ser Ile Ala     210 215 220 Met Gly Ala Ser Arg Ile Gly His Gly Ile Met Thr Lys Asn Leu Ser 225 230 235 240 Glu Ala Glu Gln Lys Met Met Ile Glu Lys Gln Ile Val Leu Glu Met                 245 250 255 Ala Pro Thr Ser Asn Phe Gln Thr Lys Ala Val Thr Glu Leu Ala Gln             260 265 270 Tyr Pro Phe Lys Glu Leu Tyr Asp Lys Gly Ile His Val Thr Leu Asn         275 280 285 Thr Asp Asn Arg Met Val Ser Ala Thr Asn Leu Ser Lys Glu Tyr Glu     290 295 300 Lys Ile Ser Ala Trp Tyr Pro Asp Phe Ser Leu Ser Asp Phe Glu Lys 305 310 315 320 Ile Asn His Tyr Ala Ile Asp Gly Ala Phe Ile Gly Gln Glu Glu Lys                 325 330 335 Glu Glu Leu His Gln Arg Phe Thr Lys Glu Tyr Lys Lys Ile Ser Glu             340 345 350 <210> 4 <211> 705 <212> DNA <213> Artificial Sequence <220> <223> Purine nucleoside phosphorylase derived from Bacillus          stearothermophilus <400> 4 atggcgttc atattggtgc aaaagaacac gagattgcag ataaaatttt gcttccagga 60 gatccacttc gcgcaaaata tatcgctgaa acgtttttag agggagctac ttgctataat 120 caagttcgcg gtatgttagg atttacaggt acatataaag gccatcgtat ttccgttcaa 180 ggaacaggta tgggtgtacc atctatttct atttatatta cagaacttat gcaaagctac 240 aacgttcaaa cattaattcg cgtcggaaca tgtggtgcta ttcaaaaaga tgtaaaagtt 300 cgtgatgtca ttttagcgat gacatcgtca accgattccc aaatgaatcg catgacgttc 360 ggaggaattg attacgctcc gacagctaac tttgacttgt taaaaacagc gtacgaaatt 420 ggaaaagaaa aaggattaca actaaaagtt ggcagtgtat ttacagctga tatgttttat 480 aatgaaaatg cacaatttga aaaactggca cgatacggtg tactggctgt agagatggaa 540 acaacagcgc tttatacatt agccgctaaa tttggcagaa aagcattatc ggtattaaca 600 gtaagcgatc acattttaac aggggaagag acaacggctg aagaccgcca aacaacattt 660 aacgaaatga tcgaagtcgc tcttgaaaca gcgattcgcc aataa 705 <210> 5 <211> 1302 <212> DNA <213> Artificial Sequence <220> <223> Pyrimidine nucleoside phosphorylase derived from Bacillus          stearothermophilus <400> 5 atgagaatgg tcgatttaat tgagaaaaaa cgtgatggtc atgcgttaac gaaagaagaa 60 attcagttta ttattgaagg ttacacaaaa ggcgatattc ctgattatca aatgagcgca 120 ttagcgatgg cgattttttt ccgcggcatg aatgaagaag agacagcgga attgacgatg 180 gcgatggtgc attcaggcga tacgatcgac ctttcgcgaa ttgaaggaat taaagtagac 240 aaacattcaa cgggcggagt gggcgataca acaacgttag tgcttggccc tcttgtcgcc 300 tccgtcggtg ttccggttgc gaaaatgtct gggcgcggcc ttggacatac gggtggaacg 360 atcgacaaac tagaatcggt gccaggtttt cacgttgaaa ttacgaacga tgaatttatc 420 gatcttgtca ataaaaataa aattgccgtt gtcggtcagt ctggtaattt gacgccagcg 480 gacaaaaagt tgtatgcgct tcgtgatgtg acggcaacgg tcaatagcat tccgttaatt 540 gcctcatcga ttatgagcaa aaaaattgcc gcaggggcag atgcgatcgt acttgacgta 600 aaaacaggtg tgggcgcgtt tatgaaagat ttaaacgatg caaaagcatt agcgaaagcg 660 atggtcgata tcggaaatcg ggttgggcgt aaaacgatgg caattatttc tgatatgagc 720 cagccgcttg gttatgccat tggaaatgcg cttgaagtga aagaagcgat tgatacgtta 780 aaaggagaag gtccagaaga tttccaagag ctgtgcttag tgcttggtag ccacatggta 840 tatttagcgg aaaaagcatc ttcgcttgaa gaagctcgtc atatgttaga aaaagcgatg 900 aaagacggtt cagcccttca aacatttaaa acgttcttag ctgcgcaagg tggcgatgca 960 tctgttgtcg atgacccaag caaattgccg caagcaaaat atattattga actagaagcg 1020 aaagaagatg gatacgtatc cgaaattgtc gcggatgcgg tcggaacggc ggcgatgtgg 1080 cttggtgcag ggcgagcgac gaaagaatca acgatcgatt tagctgtcgg tctcgtgctt 1140 cgcaaaaaag tcggcgatgc ggtgaaaaaa ggtgaatcgc tcgttacaat ttacagcaac 1200 cgtgaacaag tggatgatgt aaaacaaaaa ctatatgaaa acattcgtat ttcagcaaca 1260 cctgttcaag ctccaacatt aatttacgat aaaatttcgt aa 1302 <210> 6 <211> 1059 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Adenosine deaminase derived from Lactococcus lactis <400> 6 atgaaaaaga aaagggagaa actcgtgctt aaatcagaaa ttattgctca aatgccaaaa 60 gttgaacttc attgccatct tgatggttcg cttagcttgt cagtcattaa agaactcgct 120 aaaaatgctg gaattcatat gacaatgtcc gatgaggaaa ttttagaaaa ggcacaagcc 180 ccagaaaata caaaaaattt gctagaatac ctgcaacgtt ttgatttcgt cttaccactt 240 ttgcaaactt ataagaattt agagttggct gcttatgatg ttgtcagaca ggcagcaaat 300 gataatatta aatatattga aatccgtttt gctccaagtc aacatctctt agaaaatcta 360 acccttgaag aagcagtaga ggctgtcatc gccggccttt ctcgtgcaga aaatgatttt 420 gatataagag caaatgcact cgtttgtgga ttaaaacaag aaccaattca aaaattacaa 480 aaactgctgc cactttttga taagattcct gatgaacatc ttgtcggatt tgatatggcg 540 ggtgatgaat taaattatcc acaggaaaaa tttgtagatt tgattcatga tataaaaatt 600 aagggtgtca atgtgacgct tcatgctgga gaatgtccag catgtgagaa aaatatttta 660 gattcaattg caatgggggc aagtcgcatt ggacatggaa ttatgactaa aaatctgtcc 720 gaagccgaac aaaaaatgat gattgaaaaa caaattgttt tagaaatggc tccgactagc 780 aattttcaaa caaaagcggt gacagagctt gcgcaatatc cttttaaaga actttatgac 840 aaaggaatac acgtgacatt aaataccgat aatcgaatgg tttcggctac aaatttaagc 900 aaagaatatg agaaaatctc agcttggtac ccagattttt ctctttctga ttttgaaaaa 960 ataaatcatt acgcaattga tggcgccttt attggtcaag aagaaaaaga agaacttcat 1020 caaagattta ccaaagaata caaaaaaatc tctgaataa 1059 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BPUNP forward primer <400> 7 ttaacatatg agaggatcgc atca 24 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> BPUNP reverse primer <400> 8 agctctagat tattggcgaa tcgctgtttc 30 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> BPYNP forward primer <400> 9 atatcatatg agaatggtcg atttaattga g 31 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> BPYNP reverse primer <400> 10 cccaagcttt tacgaaattt tatcgtaaat taatg 35 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> LADD forward primer <400> 11 ggatccaatg aaaagaaaag ggagaaactc 30 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> LADD reverse primer <400> 12 aagcttctct gattattcag agattttttt g 31 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> LADD forward primer (Insert) <400> 13 agatctcata tgagaggatc tcaccatcac c 31

Claims (11)

(A) Bacillus stearothermophilus-derived purine nucleoside phosphorylase comprising the amino acid sequence of SEQ ID NO: 1 and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase comprising the amino acid sequence of SEQ ID NO: 2 Amino-3'-deoxythymidine and 2,6-diaminopurine added at a concentration of 1 M or more to prepare 3'-amino-2 ', 3'-dideoxy-2,6- Producing a diaminopurine riboside; And
(b) 3'-amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside was treated with adenosine deaminase derived from lactococcus lactis to form 3'-amino-2' - &lt; / RTI &gt; dideoxy guanosine,
The step (a) is carried out at 30 to 50 ° C,
Amino-2 ', 3'-dideoxy-2,6-diaminopurine riboside (b) 3'-amino-2', 3'-dideoxy-2,6-diaminopurine riboside was treated with adenosine deaminase derived from lactocokus lactis, '- dideoxy guanosine is prepared by directly administering adenosine deaminase derived from lactococcus lactis without the purification of the reaction product of step (a), to prepare 3'-amino-2', 3'-dideoxy guanosine Way. delete delete 3. The method according to claim 1, wherein the 3'-amino-3'-deoxythymidine and the 2,6-diaminopurine of step (a) are reacted in a molar ratio of 1: ', 3'-dideoxyguanosine. delete delete The method according to claim 1, wherein the Bacillus stearothermophilus-derived purinucleoside phosphorylase, Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase, and Lactococcus lactis-derived adenosine deaminase are genes A method for producing 3'-amino-2 ', 3'-dideoxy guanosine, which is an enzyme contained in a cell body or a cell treatment product overexpressed in E. coli by recombination. The method according to claim 1, wherein after step (b) of producing 3'-amino-2 ', 3'-dideoxy guanosine,
(c) adding an alcohol and a strong base to the reactant in step (b) to remove an enzyme source of the purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, and adenosine deaminase and reaction by-products Amino-2 &apos;, 3 ' -dideoxyguanosine. &Lt; / RTI &gt; 9. The method according to claim 8, wherein the alcohol in step (c) is a lower alcohol having 1 to 4 carbon atoms, and the strong base is at least one selected from the group consisting of sodium, potassium, calcium hydroxide, , 3'-dideoxyguanosine. 9. The method according to claim 8, wherein the enzyme source of the purin nucleoside phosphorylase, the pyrimidine nucleoside phosphorylase, and the adenosine deaminase is an enzyme source contained in the microbial cells or the microbial treatment, Amino-2 ', 3 ' -dideoxyguanosine. The method according to claim 8, wherein the step (c) comprises simultaneously removing an enzyme source and a reaction by-product by adding an alcohol and a strong base to the 3'-amino-2 ', 3'-dideoxy guanosine.

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