JP2007159585A - Method for producing 2'-deoxyadenosine and 2'-deoxyguanosine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an efficient/industrial method for producing 2'-deoxyadenosine and 2'-deoxyguanosine used as a raw material for an anti-virus agent, etc., especially an anti-sense medicine. <P>SOLUTION: The method is carried out by producing the 2'-deoxyadenosine from adenine, adenosine or 5'-adenylic acid in the presence of an inorganic phosphoric acid or a salt thereof by using a bacterium of the genus Klebsiella. In the method, the 2'-deoxyguanosine is produced from guanine, guanosine or 5'-guanylic acid under the similar conditions. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  2'-deoxyadenosine and 2'-deoxyguanosine are desired to be synthesized in large quantities as raw materials for pharmaceuticals, particularly antisense pharmaceuticals that have recently become a hot topic.

As the production method of 2′-deoxyadenosine and 2′-deoxyguanosine, the yield of the chemical synthesis method is very low. Therefore, the industrial production method mainly involves extraction from a hydrolyzate of DNA (deoxyribonucleic acid). Yes.
However, in this method, the microorganism to be used has a slight activity of degrading the nucleotide of the substrate, and the substrate is partially decomposed, so that a by-product is generated in the reaction solution, and the yield is lowered due to the decomposition of the substrate. . In addition, since the activity per cell is low, there is a disadvantage that the reaction cannot be performed by adding a high concentration substrate. However, in the conventional extraction method, 2′-deoxycytidine and thymidine are contained in the hydrolyzate of DNA in addition to the target 2′-deoxyadenosine and 2′-deoxyguanosine, and the extraction process is complicated and costly. Because of the high method, development of a more efficient method has been desired.

As a method for biochemical production of 2′-deoxynucleoside using nucleoside phosphorylase of microorganism, 2′-deoxypurine nucleoside is produced as 2′-deoxyribofuranosylpurine and 2′-deoxyribofuranosylthioguanine (specifically, No. 58-63393) As 2′-deoxypyrimidine nucleosides, 2′-deoxycytidine (JP 01-060396) and thymidine (JP 01-104190) are known, but 2′-deoxy No method for producing adenosine, 2′-deoxyguanosine was known.
JP 58-63393 A Eur. J. Biochem., Vol. 51, (1975) p.253-265

  An object of the present invention is to provide an industrially inexpensive and efficient production method of 2'-deoxyadenosine and 2'-deoxyguanosine.

Therefore, the present inventor conducted extensive studies to establish an efficient method for producing 2′-deoxyadenosine and 2′-deoxyguanosine,
(1) 2'-deoxyadenosine is produced by the action of microorganisms from deoxyribose-1-phosphate or a salt thereof and adenine, adenosine or 5'-adenylic acid,
(2) 2′-deoxyadenosine is produced by the action of microorganisms from 2′-deoxyuridine or thymidine and adenine, adenosine or 5′-adenylic acid in the presence of inorganic phosphoric acid or a salt thereof,
(3) 2'-deoxyribose-1-phosphate or a salt thereof and guanine, guanosine or 5'-guanylic acid produce 2'-deoxyguanosine by the action of a microorganism,
(4) It has been found that 2′-deoxyguanosine is produced by the action of microorganisms from 2′-deoxyuridine or thymidine and guanine, guanosine or 5′-guanylic acid in the presence of inorganic phosphoric acid or a salt thereof. It came to complete.

  The bases and base derivatives to be reacted with 2′-deoxyribose-1-phosphate, 2′-deoxyuridine or thymidine are the same as the above adenine, adenosine, 5′-adenylic acid, guanine, guanosine, 5′-guanylic acid. If the enzyme reaction proceeds, the corresponding 2′-deoxynucleoside can be produced.

That is, the present invention
(1) Ability to produce 2′-deoxyadenosine from 2′-deoxyribose-1-phosphate or a salt thereof and adenine, adenosine or 5′-adenylic acid, and Achromobacter genus, Agrobacterium genus Acinetobacter spp Genus, Pseudomonas, Streptomyces, Sporosartina, Staphylococcus, Serratia, Cellulomonas, Nocardia, Bacillus, Hafnia, Vibrio, Flavobacterium, Planococcus, Brevibacterium ,, Culture of microorganisms belonging to the genus Taminobacter, Proteus, Hemophilus, Micrococcus, Mycoplana, Microbacteria, Rhizobium or Rhodococcus, microorganisms isolated from the culture, or processed microorganisms 2′-deoxyribose-1-phosphate or a salt thereof and adenine, adenosine or 5′-adenylic acid to produce 2′-deoxyadenosine, which is collected. Production method of deoxyadenosine,
(2) Ability to produce 2'-deoxyadenosine from 2'-deoxyuridine or thymidine and adenine, adenosine or 5'-adenylic acid in the presence of inorganic phosphoric acid or a salt thereof, Bacteria, Acinetobacter, Alkagenes, Arthrobacter, Aeromonas, Escherichia, Enterobacter, Erbinia, Xanthomonas, Klebsiella, Kurthia, Kluihera, Corynebacterium, Sartina, Salmonella Citrobacter, Pseudomonas, Streptomyces, Sporosartina, Staphylococcus, Serratia, Cellulomonas, Nocardia, Bacillus, Hafnia, Vibrio, Flavobacterium, Planococcus, Brem A culture of a microorganism belonging to the genus Bacteria, Protaminobacter, Proteus, Hemophilus, Micrococcus, Mycoplana, Microbacterium, Rhizobium or Rhodococcus, or a microbial cell isolated from the culture, The treated product of the microbial cells is allowed to act on 2′-deoxyuridine or thymidine and adenine, adenosine or 5′-adenylic acid in the presence of inorganic phosphoric acid or a salt thereof to produce 2′-deoxyadenosine. A method for producing 2′-deoxyadenosine, which is characterized by being collected.
(3) Ability to produce 2′-deoxyguanosine from 2′-deoxyribose-1-phosphate or a salt thereof and guanine, guanosine or 5′-guanylic acid, and a genus Achromobacter or Agrobacterium Acinetobacter spp Genus, Pseudomonas, Streptomyces, Sporosartina, Staphylococcus, Serratia, Cellulomonas, Nocardia, Bacillus, Hafnia, Vibrio, Flavobacterium, Planococcus, Brevibacterium ,, Culture of microorganisms belonging to the genus Taminobacter, Proteus, Hemophilus, Micrococcus, Mycoplana, Microbacteria, Rhizobium or Rhodococcus, microorganisms isolated from the culture, or processed microorganisms 2'-deoxyribose-1-phosphate or a salt thereof and guanine, guanosine or 5'-guanylic acid to produce 2'-deoxyguanosine, which is collected. Production method of deoxyguanosine,
(4) Ability to produce 2'-deoxyguanosine from 2'-deoxyuridine or thymidine and guanine, guanosine or 5'-guanylic acid in the presence of inorganic phosphoric acid or a salt thereof, Bacteria, Acinetobacter, Alkagenes, Arthrobacter, Aeromonas, Escherichia, Enterobacter, Erbinia, Xanthomonas, Klebsiella, Kurthia, Kluihera, Corynebacterium, Sartina, Salmonella Citrobacter, Pseudomonas, Streptomyces, Sporosartina, Staphylococcus, Serratia, Cellulomonas, Nocardia, Bacillus, Hafnia, Vibrio, Flavobacterium, Planococcus, Brem A culture of a microorganism belonging to the genus Bacteria, Protaminobacter, Proteus, Hemophilus, Micrococcus, Mycoplana, Microbacterium, Rhizobium or Rhodococcus, or a microbial cell isolated from the culture, The treated product of the microbial cells is allowed to act on 2′-deoxyuridine or thymidine and guanine, guanosine or 5′-guanylic acid in the presence of inorganic phosphoric acid or a salt thereof to produce 2′-deoxyguanosine, The present invention relates to a method for producing 2′-deoxyguanosine, which is characterized by being collected.

  According to the present invention, 2'-deoxyadenosine and 2'-deoxyguanosine can be produced inexpensively and efficiently industrially.

  Microorganisms used in the present invention are Achromobacter genus, Agrobacterium genus, Acinetobacter genus, Alkagenes genus, Arthrobacter genus, Aeromonas genus, Escherichia genus, Enterobacter genus, Erbinia genus, Xanthomonas genus, Klebsiella genus, Kurthia genus , Kluyhera, Corynebacterium, Sartina, Salmonella, Citrobacter, Pseudomonas, Streptomyces, Sporosartina, Staphylococcus, Serratia, Cellulomonas, Nocardia, Bacteria, Bacillus , Hafnia, Vibrio, Flavobacterium, Planococcus, Brevibacterium, Protaminobacter, Proteus, Hemophilus, Micrococcus, Mycoplana, Microbacteria Belonging to the genus, Rhizobium or Rhodococcus, the ability to produce 2'-deoxyadenosine from 2'-deoxyribose-1-phosphate or a salt thereof and adenine, adenosine or 5'-adenylic acid, inorganic phosphate or its The ability to produce 2'-deoxyadenosine from adenine, adenosine or 5'-adenylic acid in the presence of a salt with 2'-deoxyuridine or thymidine, 2'-deoxyribose-1-phosphate or a salt thereof, guanine, Ability to produce 2'-deoxyguanosine from guanosine or 5'-guanylic acid or 2'-deoxyuridine or thymidine in the presence of inorganic phosphate or salt thereof and 2'-deoxy from guanine, guanosine or 5'-guanylic acid With the ability to produce guanosine Both the well but may be Re, but can be specifically exemplified microorganisms below.

Achromobacter viscosus ATCC 12448
Agrobacterium tumefaciens ATCC 4720
Acinetobacter johnsonii ATCC 9036
Alcaligenes faecalis subsp. Faecalis ATCC 8750
Arthrobacter oxydans ATCC 14358
Aeromonas salmonicida (Aeromonas salmonicida subsp. Salmonicida) ATCC 14174
Escherichia coli ATCC 10798
Enterobacter cloacae ATCC 7256
Erwinia herbicola ATCC 1453
Xanthomonas citri AJ 2785 (FERM P-3396)
Klebsiella pneumoniae IFO 3321
Kurthia zopfii ATCC 6900
Kluyvera citrophila AJ 2626 (FERM P-8193)
Corynebacterium acetoacidophilum ATCC 21407
Sartina lutea AJ 1218 (FERM P-7400)
Salmonella typhimurium AJ 2636 (FERM P-3753)
Citrobacter freundi ATCC 8090
Pseudomonas diminuta ATCC 11568
Streptomyces tanashiens ATCC 15238
Sporosarcina ureae IFO 12698
Staphyrococcus epidermidis ATCC 155
Serratia marcescens ATCC 14226
Cellulomonas flavigera ATCC 486
Nocardia asteroides ATCC 19247
Bacillus subtilis ATCC 6633
Hafnia alvei ATCC 9760
Vibrio metschnikovii ATCC 7708
Flavobacterium breve ATCC 14234
Planococcus eucinatus AJ 1656 (FERM P-9133)
Brevibacterium pusillum ATCC 19096
Protaminobacter alboflavus ATCC 8458
Proteus rettegeri AJ 2770 (FERM BP-941)
Haemophilus influenzae ATCC 9134
Micrococcus luteus ATCC 4698
Mycoplana dimorpha ATCC 4279
Microbacterium lacticum ATCC 8180
Rhizobium melilotti AJ 2823 (FERM P-8197)
Rhodococcus rhodochraus ATCC 19149

Among the above strains, Xanthomonas citri AJ 2785 (FERM P-3396)
International Depositary Authority: The FERM P-3396 deposit number deposited on January 27, 1975, at the Institute of Microbial Industrial Technology, Ministry of International Trade and Industry (now the Institute of Biotechnology, Institute of Industrial Technology, Ministry of International Trade and Industry) Kluyvera citrophila AJ 2626 (FERM P-8193) is a strain of accession number FERM P-8193 deposited on April 23, 1985 with the same depositary authority. lutea) AJ 1218 (FERM P-7400) is a strain of accession number FERM P-7400 deposited on January 20, 1984 with the same depositary authority, Salmonella typhimurium AJ 2636 (FERM P- 3753) is a strain with the deposit number FERM P-3753 deposited on October 6, 1976, and the planococcus eucinatus AJ 1656 (FERM P-9133) Accession number FERM P deposited on January 19, 1987 Proteus rettegeri AJ 2770 (FERM BP-941) was deposited with the depository authority on April 25, 1985, and was based on the Budapest Treaty on November 28, 1985 Rhizobium melilotti AJ 2823 (FERM P-8197) is a strain with the accession number FERM BP-941 transferred to the international deposit, and the accession number deposited on April 25, 1985 with the depositary authority. It is a strain of FERM P-8197.

As a method for causing 2′-deoxyadenosine or 2′-deoxyguanosine using these microorganisms, a culture method in which a substrate is added during the culture of the microorganism may be used, or the cultured cells or this treated product may be used as a substrate. You may use the static cell method made to act on.
When using the culture method, a normal medium containing a carbon source, a nitrogen source, a phosphorus source, an S source, inorganic ions, etc., and if necessary, vitamins and organic nitrogen sources may be used.
Examples of carbon sources include carbohydrates such as glucose, alcohols such as glycerol, organic acids such as acetic acid, and nitrogen sources such as ammonia gas, aqueous ammonia, ammonium salts, nitric acid and salts thereof, and phosphorus sources. Inorganic phosphoric acid and its salts such as monopotassium phosphate, magnesium sulfate as the S source, magnesium ions, potassium ions, iron ions, manganese ions, etc. are used as needed as inorganic ions . As organic nutrient sources, vitamins, amino acids and the like, and yeast extract, peptone, meat extract, cone-prica, casein degradation products and the like containing these are suitably used. The culture conditions are not particularly limited. For example, the culture may be performed for about 12 to 72 hours while appropriately limiting the pH and temperature within the range of pH 5 to 8 and temperature 25 to 40 ° C under aerobic conditions.

In particular,
(1) When 2'-deoxyribose-1-phosphate is produced from 2'-deoxyribose-1-phosphate, 2'-deoxyribose-1-phosphate and adenine or adenosine or 5'- Add adenylic acid as appropriate,
(2) When 2'-deoxyguanosine 2'-deoxyguanosine is produced from 2'-deoxyribose-1-phosphate, 2'-deoxyribose-1-phosphate and guanine or Add guanosine or 5'-guanylic acid as appropriate,
(3) When producing 2′-deoxyadenosine from 2′-deoxyuridine or thymidine, 2′-deoxyuridine or thymidine and adenine, adenosine, or 5′-adenylic acid are appropriately added to the medium.
Or (4) When 2′-deoxyguanosine is produced from 2′-deoxyuridine or thymidine, 2′-deoxyuridine or thymidine and guanine, guanosine or 5′-guanylic acid are appropriately added to the medium. Just do it. The substrate may be added at the beginning of the culture or during the culture.

  As an enzyme source in the case of using the static cell method, the culture solution obtained by the above method cultured in the above basic medium can be used as it is, or a washed cell can be used, and a cell-treated product can also be used. Examples of treated cells include acetone-dried cells, ground cells, treated cells such as ultrasonic waves or dynomill or French press, cells contacted with a surfactant or toluene, lysozyme, protease, etc. Any of the cells treated with this enzyme, the protein fraction extracted from the cells and then separated by dialysis, etc., the purified enzyme having the enzymatic activity of this reaction, and the immobilized cells or the treated product can be used. .

In particular,
(1) When 2'-deoxyribose-1-phosphate is produced from 2'-deoxyribose-1-phosphate, it contains 2'-deoxyribose-1-phosphate and adenine or adenosine, 5'-adenylic acid The microbial cells or processed product thereof in an aqueous solution,
(2) When 2'-deoxyguanosine 2'-deoxyguanosine is produced from 2'-deoxyribose-1-phosphate, 2'-deoxyribose-1-phosphate and guanine, guanosine or 5 ' -An aqueous solution containing guanylic acid, the microbial cell or a treated product thereof,
(3) When 2'-deoxyadenosine is produced from 2'-deoxyuridine or thymidine, the microbial cell or treatment thereof in an aqueous solution containing 2'-deoxyuridine or thymidine and adenine, adenosine or 5'-adenylic acid object,
Or (4) when 2'-deoxyguanosine is produced from 2'-deoxyuridine or thymidine, the microbial cell or the microbial cell in an aqueous solution containing 2'-deoxyuridine or thymidine and guanine, guanosine or 5'-guanylic acid What is necessary is just to react by adding the processed material and adding suitably.

The reaction is usually at a temperature of 20 to 80 ° C., preferably 40 to 70 ° C., and a pH of 3 to 11 and preferably a pH of 4 to 10 gives good results. Either a stationary method or a stirring method can be employed for the reaction. The reaction time varies depending on conditions such as the activity of the enzyme used and the substrate concentration, but it may be held for 10 minutes to 10 days.
In order to collect and separate the 2′-deoxynucleoside thus produced from the reaction-terminated mixture, a method using a synthetic adsorption resin or other ordinary collection and separation methods can be employed. Further, 2′-deoxyguanosine and 2′-deoxyadenosine may be quantified by a method using high performance liquid chromatography.
Hereinafter, the present invention will be described in detail with reference to examples.

(Example 1)
50 ml of nutrient medium (pH 7.0) containing 5 g / l of yeast extract, 10 g / l of meat extract, 10 g / l of peptone and 5 g / l of NaCl is placed in a Sakaguchi flask (500 ml) at 120 ° C.
Sterilized by heating for 20 minutes. Each of the microorganisms shown in Table 1 was cultivated on a bouillon agar medium at 30 ° C. for 16 hours and inoculated with one platinum loop, and cultured at 30 ° C. for 18 hours with shaking. After separating the cells from the culture broth by centrifugation, 50 mM Tris buffer
Washed cells were prepared by washing with (pH 7.2) and further centrifuging.
The washed cells were added to 10 ml of 50 mM Tris buffer (pH 7.2) containing 100 mM 2′-deoxyribose-1-phosphate and 100 mM adenine, or 100 mM guanosine. / l and added to react at 60 ° C for 2 hours. The concentration of 2′-deoxyadenosine or 2′-deoxyguanosine produced in the reaction solution was measured using high performance liquid chromatography. The results are shown in Table 1.

(Example 2)
50 ml of nutrient medium (pH 7.0) containing 5 g / l of yeast extract, 10 g / l of meat extract, 10 g / l of peptone and 5 g / l of NaCl is placed in a Sakaguchi flask (500 ml) at 120 ° C.
Sterilized by heating for 20 minutes. To this, one platinum loop of each of the microorganisms shown in Table 2 was cultivated on a bouillon agar medium at 30 ° C. for 16 hours, and cultured with shaking at 30 ° C. for 18 hours. The bacterial cells were separated from the obtained culture broth by centrifugation, washed with 50 mM phosphate buffer (pH 7.0), and further centrifuged to prepare washed bacterial cells.
Add the washed cells to 10 ml of 50 mM phosphate buffer (pH 7.0) containing 100 mM 2'-deoxyuridine and 100 mM adenine or guanosine to 50 g / l. And reacted at 60 ° C. for 2 hours.
The concentration of 2′-deoxyadenosine or 2′-deoxyguanosine produced in the reaction solution was measured using high performance liquid chromatography, and the results are shown in Table 2.

(Example 3)
50 ml of nutrient medium (pH 7.0) containing 5 g / l of yeast extract, 10 g / l of meat extract, 10 g / l of peptone and 5 g / l of NaCl is placed in a Sakaguchi flask (500 ml) at 120 ° C.
Sterilized by heating for 20 minutes. This was inoculated with 1 platinum ear of Klebsiella pneumoniae IFO 3321 cultured on a bouillon agar medium at 30 ° C. for 16 hours, and cultured with shaking at 30 ° C. for 18 hours. The bacterial cells were separated from the obtained culture broth by centrifugation, washed with 50 mM phosphate buffer (pH 7.0), and further centrifuged to prepare washed bacterial cells.
The washed cells were washed with 50 mM phosphate buffer (pH) containing 100 mM 2'-deoxyuridine and 100 mM adenine, or adenosine, 5'-adenylic acid, guanine, or guanosine, or 5'-guanylic acid. 7.0)
The washed cells were added to 10 ml so as to be 50 g / l and reacted at 60 ° C. for 2 hours.
The concentration of 2′-deoxyadenosine or 2′-deoxyguanosine produced in the reaction solution was measured using high performance liquid chromatography, and the results are shown in Table 3.

(Example 4)
50 ml of nutrient medium (pH 7.0) containing 5 g / l of yeast extract, 10 g / l of meat extract, 10 g / l of peptone and 5 g / l of NaCl is placed in a Sakaguchi flask (500 ml) at 120 ° C.
Sterilized by heating for 20 minutes. This was inoculated with 1 platinum ear of Klebsiella pneumoniae IFO 3321 cultured on a bouillon agar medium at 30 ° C. for 16 hours, and cultured with shaking at 30 ° C. for 18 hours. The bacterial cells were separated from the obtained culture broth by centrifugation, washed with 50 mM phosphate buffer (pH 7.0), and further centrifuged to prepare washed bacterial cells.
The washed cells were washed with 50 mM phosphate buffer (pH 7.0) containing 100 mM thymidine and 100 mM adenine, adenosine, 5′-adenylic acid, guanine, guanosine, or 5′-guanylic acid. The above washed cells were added to ml at 50 g / l and reacted at 60 ° C. for 2 hours.
The concentration of 2′-deoxyadenosine or 2′-deoxyguanosine produced in the reaction solution was measured using high performance liquid chromatography, and the results are shown in Table 4.

Claims (2)

A culture of a microorganism belonging to the genus Klebsiella having the ability to produce 2'-deoxyadenosine from thymidine and adenine, adenosine or 5'-adenylic acid in the presence of inorganic phosphate or a salt thereof, and a microorganism isolated from the culture A cell or a treated product of the microorganism is reacted with thymidine and adenine, adenosine, or 5'-adenylic acid in the presence of inorganic phosphoric acid or a salt thereof to produce 2'-deoxyadenosine, which is collected. A process for producing 2'-deoxyadenosine, characterized in that Culture of microorganism belonging to genus Klebsiella having ability to produce 2'-deoxyguanosine from thymidine and guanine, guanosine or 5'-guanylic acid in the presence of inorganic phosphoric acid or a salt thereof, microorganism isolated from the culture The bacterial cell or the treated microorganism is reacted with thymidine and guanine, guanosine, or 5'-guanylic acid in the presence of inorganic phosphoric acid or a salt thereof to produce 2'-deoxyguanosine, which is collected A process for producing 2'-deoxyguanosine,