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WO2017164615A1 - Procédé de préparation de 3'-amino-2',3'-didésoxyadénosine au moyen d'une nucléoside phosphorylase dérivée de bacillus - Google Patents

Procédé de préparation de 3'-amino-2',3'-didésoxyadénosine au moyen d'une nucléoside phosphorylase dérivée de bacillus Download PDF

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WO2017164615A1
WO2017164615A1 PCT/KR2017/003014 KR2017003014W WO2017164615A1 WO 2017164615 A1 WO2017164615 A1 WO 2017164615A1 KR 2017003014 W KR2017003014 W KR 2017003014W WO 2017164615 A1 WO2017164615 A1 WO 2017164615A1
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amino
dideoxyadenosine
nucleoside phosphorylase
derived
bacillus stearothermophilus
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Kap Soo Noh
Byung Kyun Kim
Kang Hyun Choi
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ST Pharm Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02001—Purine-nucleoside phosphorylase (2.4.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02002—Pyrimidine-nucleoside phosphorylase (2.4.2.2)

Definitions

  • the present invention relates to a method for preparing 3'-amino-2',3'-dideoxyadenosine using Bacillus-derived nucleoside phosphorylase.
  • oligonucleotide and oligonucleotide analogue drugs based on binding to specific nucleic acid sequences or proteins have been conducted. Particularly, oligonucleotide analogue drugs have been studied so that their resistance to nuclease and their binding force and specificity for other substances can be improved.
  • an oligonucleotide having a N3' P5' phosphoramidate bind was reported to have resistance to nuclease by specific complementary binding to a target gene sequence in vivo and ex vivo (J. K. Chen et. al., Nucleic Acids Res., 23, 2661-2668 (1994); C. Escude et. al., Proc. Natl. Acad. Sci. USA, 93, 4365-4369 (1996); S. M. Gryaznov et. al. Nucleic Acids Res., 24, 1508-0514 (1996); C. Giovannangeli et. al., Proc. Natl. Acad. Sci.
  • 3'-azido-3'-deoxythymidine has been used as a precursor for the research development of chemical and enzymatic method for preparing of 3'-amino-2',3'-dideoxyadenosine(ADA).
  • 3'-azido-3'-deoxythymidine (AZT) is an important pharmaceutical raw material found to have anti-human immunodeficiency virus activity, and is prepared using thymidine (TMD) as a raw material (N. Miller et al., J. Org. Chem., 29, 1772-1776 (1964), Horwitz et. al., J. Org. Chem., 29, 2076-2078 (1964)). It is industrially produced in large amounts and is easily available.
  • the synthesized product was converted to 9-(3'-azido-2',3'-dideoxy-D-ribofuranosyl)adenine by a deacylation reaction using an alcohol including a strong base, and then the remaining base was removed using ion exchange resin, and 9-(3'-azido-2',3'-dideoxy- ⁇ -D-ribofuranosyl)adenine was recovered using a silica gel column. It was reported that 3'-amino-2',3'-dideoxyadenosine could be synthesized by reducing the azide moiety of the cam moiety using triphenylphosphine to the recovered compound(M. Imazawa and F. Eckstein, J. Org.
  • Zaitseva et. al. (G. V. Zaitseva et. al., Nucleosides & Nucleotides, 13(1-3), 819-834(1994)) reported that 110 mg of 3'-amino-2',3'-dideoxyadenosine was produced in a molar yield of 46% by adding 477 mg (10 mM) of 3'-amino-3'-deoxythymidine, 800 mg (30 mM) of adenine, and 3 g of dried Escherichia coli BM-11 pretreated with glutaraldehyde, to 200 ml of a reaction solution, and reacting the mixture in 5 mM of phosphate buffer (pH 6.75) at 50°C for 24 hours, followed by purification using ion exchange resin or the like.
  • phosphate buffer pH 6.75
  • 3'-amino-2',3'-dideoxyadenosine could be synthesized by the action of thymidine phosphorylase (EC2.4.2.4), uridine phosphorylase (EC2.4.2.3) or purine nucleoside phosphorylase (PUNP, EC2.4.2.1), which is present in the E. coli strain, but the specific yield and synthesis of 3'-amino-2',3'-dideoxyadenosine were not reported.
  • US Patent Publication No. 2007/0065922 discloses the results of performing an enzymatic reaction using selected Escherichia coli 1K/1T showing high activities of thymidine phosphorylase and purine nucleoside phosphorylase.
  • the amount of microbial cells used to convert 10 mmol (2.42 g) of 3'-amino-3'-deoxythymidine reaches 0.6-15 g (on a dry cell basis), and when such a large amount of microbial cells are used, industrialization is impossible due to the raw material cost for preparing the microbial cells, the process time required to remove the microbial cells after completion of bioconversion, and a decrease in the yield.
  • the efficiency of equipment is reduced because the concentration of the main raw material 3'-amino-3'-deoxythymidine is low (10 mM to 100 mM).
  • the molar ratio between reaction substrates shows a difference of 2-3 times in order to increase the transglycosylation yield, and thus a large amount of unreacted materials remain after completion of enzymatic conversion to impose a burden on subsequent purification and to cause the material cost in industrial terms, making it unsuitable for mass production.
  • the purification method using ion exchange resin after the bioconversion reaction has problems in that a separate resin tower is required in process design and in that the addition of adsorption and desorption processes and a process for concentration of product fractions increases the process time and the production cost.
  • the present invention provides a method for preparing 3'-amino-2',3'-dideoxyadenosine, comprising a step of preparing 3'-amino-2',3'-dideoxyadenosine by treating 3'-amino-3'-deoxythymidine and adenine with Bacillus stearothermophilus-derived purine nucleoside phosphorylase and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase.
  • the present invention also provides a method for preparing 3'-amino-2',3'-dideoxyadenosine, further comprising, after the enzymatic reaction, a step of removing the sources of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase enzymes, and reaction byproducts by adding an alcohol and a strong base to the enzymatic reaction mixture of preparing 3'-amino-2',3'-dideoxyadenosine.
  • the present inventors have made extensive efforts to develop a method for industrial production of a large amount of 3'-amino-2',3'-dideoxyadenosine, and as a result, have found that when Bacillus stearothermophilus-derived purine nucleoside phosphorylase and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase are used, a high purity of 3'-amino-2',3'-dideoxyadenosine can be produced in a higher yield compared to when a conventional known method is used, thereby completing the present invention.
  • the present invention provides a method for preparing 3'-amino-2',3'-dideoxyadenosine, comprising a step of producing 3'-amino-2',3'-dideoxyadenosine by treating 3'-amino-3'-deoxythymidine and adenine with Bacillus stearothermophilus-derived purine nucleoside phosphorylase and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase.
  • 3'-amino-2',3'-dideoxyadenosine is a compound having a structure of the following formula 1:
  • 3'-amino-3'-deoxythymidine is a compound having a structure of the following formula 2:
  • the adenine is a compound having a structure of the following formula 3:
  • thymine is a compound having a structure of the following formula 4:
  • nucleoside phosphorylase refers to an enzyme that causes phosphorolysis of a N-glycosidic linkage of nucleoside in the presence of phosphate, and catalyzes a reaction represented by the following equation:
  • the method for preparing 3'-amino-2',3'-dideoxyadenosine according to the present invention exhibits a high rate of conversion to 3'-amino-2',3'-dideoxyadenosine by use of Bacillus stearothermophilus-derived nucleoside phosphorylases, and thus can efficiently produce a large amount of 3'-amino-2',3'-dideoxyadenosine.
  • the method according to the present invention shows a significantly high ability to produce 3'-amino-2',3'-dideoxyadenosine, compared to a preparation method using the purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase derived from other strains, or a preparation method using other nucleoside phosphorylases.
  • bioconversion process which reacts 3'-amino-3'-deoxythymidine and adenine with Bacillus stearothermophilus-derived purine nucleoside phosphorylase and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase, is explained by the following two-step transglycosylation process:
  • pyrimidine nucleoside phosphorylase substitutes 3'-amino-3'-deoxythymidine with 3-amino-2,3-dideoxyribose-1-phosphate, and 3-amino-2,3-dideoxyribose-1-phosphate is converted to 3'-amino-2',3'-dideoxyadenosine by purine nucleoside phosphorylase.
  • pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase are Bacillus stearothermophilus-derived purine nucleoside phosphorylase and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase, respectively.
  • the Bacillus stearothermophilus is Bacillus stearothermophilus TH6-2 (FERM BP-2758).
  • the molecular biological characteristics and/or amino acid sequences of the above-described Bacillus stearothermophilus-derived purine nucleoside phosphorylase and/or Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase can be analyzed, and based on the analysis, the genes of the proteins can be obtained from the strain.
  • a recombinant plasmid having inserted therein the gene and a control region necessary for expression can be constructed and introduced into any host, thereby constructing a genetic recombinant strain having the protein expressed therein.
  • a genetic recombinant strain obtained by introducing the Bacillus stearothermophilus-derived purine nucleoside phosphorylase and/or Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase gene into any strain also falls within the scope of the present invention.
  • Bacillus stearothermophilus-derived purine nucleoside phosphorylase comprises a purine nucleoside phosphorylase of SEQ ID NO: 1 or its active fragment
  • Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase comprises a pyrimidine nucleoside phosphorylase of SEQ ID NO: 2 or its active fragment.
  • Bacillus stearothermophilus-derived purine nucleoside phosphorylase may be synthesized from a nucleotide sequence of SEQ ID NO: 3.
  • Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase may be synthesized from a nucleotide sequence of SEQ ID NO: 4.
  • the above-described recombinant plasmid refers to a genetic construct including essential regulatory elements operably linked to express a gene insert.
  • the control region necessary for expression includes a promoter sequence (including a operator sequence to control transcription), a ribosome-binding sequence (a SD sequence), a transcription termination sequence and the like, and also includes a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein.
  • the promoter sequence may be a trp promoter of a tryptophan operon derived from E. coli, a lac promoter of a lactose operon, a promoter derived from ⁇ phage, or a gluconic acid synthase promoter (gnt) derived from Bacillus subtilis, an alkaline protease promoter (apr), a neutral protease promoter (npr), and an ⁇ -amylase promoter (amy). Sequences specifically designed and modified, such as a tac promoter, may also be used.
  • ribosome-binding sequence examples include those sequences derived from E. coli or B. subtilis, but are not particularly limited as long as they function within a desirable host such as E. coli, B. subtilis or the like.
  • a consensus sequence where a sequence of 4 or more consecutive bases is complementary to the 3'-terminal region of a 16S ribosomal RNA may be prepared by DNA synthesis and used.
  • the transcription termination sequence is not essential, but, if necessary, ones independent of the ⁇ factor, such as a lipoprotein terminator, a trp operon terminator and the like can be used.
  • Sequences on a recombinant plasmid having these control regions are preferably arranged in the order of a promoter sequence, a ribosome binding sequence, nucleoside phosphorylase-encoding genes and a transcription termination sequence, from the 5'-terminal upstream.
  • Examples of the plasmid include, but are not limited to, pFRPT (Korean Patent No. 10-0449639), pBR322, pUC18, Bluescript II SK(+), pKK223-3 and pSC101, which has a region capable of self-replication in E. coli, or pUB110, pTZ4, pC194, ⁇ 11 and ⁇ -105, which have a region capable of self-replication in B. subtilis.
  • examples of the plasmid capable of self-replication in two or more kinds of hosts include pHV14, TRp7, YEp7, pBS7 and the like.
  • Examples of any host as described above include, but are not limited to, Escherichia coli, Bacillus sp. strains such as Bacillus subtilis, etc. Preferably, Escherichia coli which is industrially easily available may be used. In an embodiment of the present invention, the host is Escherichia coli JM109.
  • usable forms of the Bacillus stearothermophilus-derived purine nucleoside phosphorylase and/or pyrimidine nucleoside phosphorylase include all enzymes themselves, microbial cells having enzymatic activity, treated microbial cells, or immobilized materials thereof, and may be used to perform the reactions.
  • the microbial cells may be wet microbial cells isolated by centrifugation, or freeze-dried microbial cells.
  • treated microbial cells is meant to include, for example, acetone-dried microbial cells, or microbial cell lysates prepared by mechanical disruption, ultrasonic disruption, freezing-thawing treatment, pressurization-depressurization treatment, osmotic treatment, self-digestion, cell wall degradation, surfactant treatment, etc.
  • the treated microbial cells if necessary, include treated microbial cells repeatedly purified by ammonium sulfate precipitation, or acetone precipitation, column chromatography.
  • the present inventors inserted the Bacillus stearothermophilus-derived purine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase gene into the E. coli expression vector pFRPT (Korean Patent No. 0449639), and introduced the expression vector into E. coli JM109, thereby constructing genetic recombinant strains, pFRPT-BPUNP/JM109 and pFRPT-BPYNP/JM109.
  • the step of producing 3'-amino-2',3'-dideoxyadenosine by treating 3'-amino-3'-deoxythymidine and adenine with Bacillus stearothermophilus-derived purine nucleoside phosphorylase and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase can efficiently produce 3'-amino-2',3'-dideoxyadenosine by reactions under the following conditions.
  • Bacillus stearothermophilus-derived purine nucleoside phosphorylase and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase were calculated as follows:
  • the optimal enzymatic using ratio between Bacillus stearothermophilus-derived purine nucleoside phosphorylase and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase which are used in the present invention, but are not limited to, is an enzymatic activity ratio of 1 U (Bacillus stearothermophilus-derived purine nucleoside phosphorylase) : 1-3 U (pyrimidine nucleoside phosphorylase activity), preferably 1 U: 2 U.
  • the reaction pH in the enzymatic reaction by treating 3'-amino-3'-deoxythymidine and adenine with Bacillus stearothermophilus-derived purine nucleoside phosphorylase and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase may be pH 7.5 to 9.5, preferably pH 8.0 to 9.0, more preferably pH 8.5 to 9.0.
  • the reaction temperature in the step of preparing 3'-amino-2',3'-dideoxyadenosine by treating 3'-amino-3'-deoxythymidine and adenine with Bacillus stearothermophilus-derived purine nucleoside phosphorylase and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase may be below 50°C, preferably 30 to 40°C, more preferably about 40°C.
  • the reaction temperature may preferably be set at about 40°C in order to suppress the above-described phenomenon.
  • the reaction time in the step of preparing 3'-amino-2',3'-dideoxyadenosine by treating 3'-amino-3'-deoxythymidine and adenine with Bacillus stearothermophilus-derived purine nucleoside phosphorylase and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase may vary depending on the amount of enzymes treated, but is 48-96 hours, preferably 48-60 hours.
  • the reaction in the step of preparing 3'-amino-2',3'-dideoxyadenosine by treating 3'-amino-3'-deoxythymidine and adenine with Bacillus stearothermophilus-derived purine nucleoside phosphorylase and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase may be performed in the presence of phosphate or its salt.
  • phosphate or its salt may preferably be sodium dihydrogen phosphate, and may be contained in the reaction solution at a low concentration of 3-10 mM, preferably 3-5 mM.
  • the reaction of 3'-amino-3'-deoxythymidine and adenine is preferably performed at a substrate concentration of 1 M or higher. Namely, the reaction is easily carried out at a high concentration substrate to provide a preparation method suitable for the development of industrial preparation processes.
  • 3'-amino-3'-deoxythymidine and adenine are preferably used at a molar ratio of 1:1.
  • the substrates are used at a molar ratio of 1:1, there are advantages in that the waste of raw materials can be reduced and in that purification in a subsequent process can be more easily performed. This reduces the waste of raw materials and facilitates purification in a subsequent process, unlike conventional known methods in which a conversion reaction is performed in a state in which the ratio of equivalents of a specific substrate is increased.
  • the present invention also provides a method for preparing 3'-amino-2',3'-dideoxyadenosine, further comprising, after the step of preparing 3'-amino-2',3'-dideoxyadenosine, a step of removing purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase enzyme sources and reaction byproducts by adding an alcohol and a strong base to the enzymatic reaction mixture of preparing 3'-amino-2',3'-dideoxyadenosine.
  • the method for preparing 3'-amino-2',3'-dideoxyadenosine according to the present invention further comprises the above step, it can produce 3'-amino-2',3'-dideoxyadenosine in high yield without having to perform purification by ion exchange resin, thereby solving the problem in that the process time is increased by adsorption, desorption and fraction concentration in methods that use ion exchange resin or the like.
  • the method of the present invention is suitable for industrial mass production.
  • the step of removing purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase enzyme sources and reaction byproducts by adding an alcohol and a strong base to the enzymatic reaction mixture of preparing 3'-amino-2',3'-dideoxyadenosine makes it possible to efficiently produce 3'-amino-2',3'-dideoxyadenosine by a reaction under the following conditions, and makes it possible to purify 3'-amino-2',3'-dideoxyadenosine with high purity.
  • the step of removing purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase enzyme sources and reaction byproducts by adding an alcohol and a strong base to the enzymatic reaction mixture of preparing 3'-amino-2',3'-dideoxyadenosine is a step of simultaneously removing unreacted substrates remaining after the reaction with Bacillus stearothermophilus-derived purine nucleoside phosphorylase and Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase enzyme sources and the major byproduct thymine produced by the reaction.
  • the step of removing the enzyme sources and the reaction byproducts by adding an alcohol and a strong base to the enzymatic reaction mixture of preparing 3'-amino-2',3'-dideoxyadenosine is a process of purifying the desired material from the bioconversion reaction solution.
  • removal of microbial cells used as the enzyme sources may first be performed.
  • the microbial cells may generally be filtered out using ultrafiltration (UF), membrane filter (MF) filtration, continuous centrifugation, and diatomaceous earth filtration.
  • UF ultrafiltration
  • MF membrane filter
  • diatomaceous earth filtration is used.
  • the filtration is preferably performed in a state in which materials except for the enzyme sources (preferably microbial cells) are dissolved in a solvent.
  • a strong base is preferably added after enzymatic conversion to completely dissolve nucleosides and bases except for microbial cells, followed by filtration process of the microbial cells.
  • a process of removing byproducts, including thymine may be performed after a process of removing enzyme sources (preferably microbial cells) by filtration.
  • a process of removing the major byproduct thymine may be performed.
  • thymine Unlike nucleosides and other bases, thymine was found to have a very low solubility in a solution of a strong base in an alcohol, and thus when the solution from which the enzyme sources (preferably microbial cells) were removed by filtration is treated with an alcoholic suspension of a base, byproducts such as thymine can be removed.
  • the reactant of the step of preparing 3'-amino-2',3'-dideoxyadenosine may be treated with a solution of a strong base in an alcohol together with diatomaceous earth to thereby remove simultaneously remove the enzyme sources (preferably microbial cells) and thymine.
  • the process of simultaneously removing the microbial cells and the enzyme sources has the advantage of simplifying the process, and also has an advantage over the process of removing only the microbial cells in that thymine captures the microbial cells to improve the filtration property to thereby reduce the separation process time.
  • the strong base that is used in the present invention may be any one or more selected from among sodium hydroxide, potassium hydroxide, calcium hydroxide, and barium hydroxide, and the alcohol that is used in the present invention may be selected from among lower alcohols having 1 to 4 carbon atoms, preferably methyl alcohol, ethyl alcohol, 1-propyl alcohol, and 2-propyl alcohol.
  • an alcoholic suspension of a strong base, which is used in the present invention may be a 0.5N solution of sodium hydroxide-methyl alcohol suspension, which is used in an amount equal to 8 to 12 times (preferably about 10 times) based on the amount of 3'-amino-3'-deoxythymidine used.
  • partial concentration may be performed.
  • a lower alcohol having 1 to 4 carbon atoms preferably methyl alcohol, ethyl alcohol, 1-propyl alcohol, and 2-propyl alcohol, may be added to the filtrate in an amount equal to 3 to 7 times (preferably 5 times) the amount of 3'-amino-3'-deoxythymidine added, after which the suspension containing the lower alcohol having 1 to 4 carbon atoms may be adjusted to a water content of 14-17%, followed by heating and cooling, thereby further removing the remaining byproduct thymine.
  • the filtrate from which thymine was removed as described above may be completely concentrated, and then 3'-amino-2',3'-dideoxyadenosine may be crystallized using an alcohol.
  • 3'-amino-2',3'-dideoxyadenosine may efficiently be crystallized.
  • the above crystallization step may be repeated in order to produce 3'-amino-2',3'-dideoxyadenosine with high purity, and a filtration process using a 0.2 ⁇ m membrane (MF) filter may be added between the crystallization steps to remove impurities incorporated during the process and microbial cell-derived water-soluble protein aggregates.
  • MF membrane
  • the method for preparing 3'-amino-2',3'-dideoxyadenosine according to the present invention shows a high rate of conversion to 3'-amino-2',3'-dideoxyadenosine by use of Bacillus stearothermophilus-derived nucleoside phosphorylases, and thus can produce 3'-amino-2',3'-dideoxyadenosine in high yield and produce 3'-amino-2',3'-dideoxyadenosine with high purity without having to perform purification by ion exchange resin. Therefore, the method of the present invention can produce a large amount of 3'-amino-2',3'-dideoxyadenosine in an economical and efficient manner.
  • FIG. 1 shows a process for preparing 3'-amino-2',3'-dideoxyadenosine according to the present invention.
  • FIG. 2 shows the structure of a pFRPT-BPUNP expression vector prepared according to the present invention.
  • FIG. 3 shows the structure of a pFRPT-BPYNP expression vector prepared according to the present invention.
  • FIG. 4 shows the results of HPLC analysis performed in each process of purifying 3'-amino-2',3'-dideoxyadenosine prepared according to the present invention.
  • Example 1 Preparation of Strain Expressing Bacillus stearothermophilus-Derived Purine Nucleoside Phosphorylase
  • a transformed E. coli strain overexpressing Bacillus stearothermophilus-derived purine nucleoside phosphorylase (BPUNP) to be used for transglycosylation in a process for preparation of 3'-amino-2',3'-dideoxyadenosine was prepared in the following manner.
  • a PCR reaction was performed in the presence of 200 ⁇ M of dNTP, 20 pmol of each primer, 1x Taq DNA polymerase buffer and 2.5 U of Taq DNA polymerase (TaKaRa Ex Taq, cat. # RR001A, TAKARA, Japan, www.takara-bio.com) for 30 cycles, each consisting of 30 sec at 94°C, 1 min at 50°C and 1 min at 72°C.
  • amplified 723 bp PCR product was confirmed by agarose gel electrophoresis, and purified using a Gel Extraction Kit(QIAquick Gel Extraction Kit, cat.
  • the purified DNA fragment was ligated into a pGEM-T easy vector (pGEM-T easy vector system II, cat. # A1380, Promega, USA, www.promega.com), and then transformed into JM109 E. coli cells (included in pGEM-T easy vector system II). From the resulting E. coli colony, a colony containing the desired plasmid was selected and named “pGEM-BPUNP/JM109”.
  • the pGEM-BPUNP/JM109 strain was inoculated into 3 ml of the medium shown in Table 2 below and was cultured overnight at 37°C, 200 rpm and pH 7, followed by centrifugation to harvest cells. Using a Plasmid purification kit(Dyne Plasmid Miniprep Kit, cat. # A510, Dynebio, Korea, www.dynebio.co.kr), pGEM-BPUNP was isolated.
  • Table 2 Components of medium for culture of pGEM-BPUNP/JM109 strain
  • pGEM-BPUNP was digested in 20 ⁇ l of a solution containing 10 U of NdeI and 10 U of XbaI, and analyzed by agarose gel electrophoresis as described above, and a 716 bp BPUNP DNA fragment was purified using a gel extraction kit.
  • the two DNA fragments obtained as described above were incubated in a solution containing ligase of 3 U (T4 DNA ligase, cat. # 2011A, TAKARA, Japan, www.takara-bio.com) and 1X ligase buffer at 16°C for 18 hours.
  • JM109 E. coli cells were transformed with the reaction solution and cultured, and from the resulting E. coli colonies, a plasmid was extracted.
  • the plasmid having the desired DNA fragment inserted therein was named “pFRPT-BPUNP”.
  • FIG. 2 schematically shows the plasmid.
  • the transformed strain obtained as described above was named “pFRPT-BPUNP/JM109”.
  • a transformed E. coli strain overexpressing Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase (BPYNP) to be used for transglycosylation in a process for preparation of 3'-amino-2',3'-dideoxyadenosine was constructed in the following manner.
  • a PCR reaction was performed in the presence of 200 ⁇ M of dNTP, 20 pmol of each primer, 1x Taq DNA polymerase buffer and 2.5 U of Taq DNA polymerase for 30 cycles, each consisting of 1 min at 94°C, 1 min at 55°C and 2 min at 72°C.
  • 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 into a pGEM-T easy vector, and then transformed into JM109 E. coli cells. Among the resulting E.
  • pGEM-BPYNP/JM109 a clone containing the desired plasmid was selected and named “pGEM-BPYNP/JM109”.
  • the pGEM-BPYNP/JM109 strain was inoculated into 3 ml of the medium shown in Table 2 above and was cultured overnight at 37°C, 200 rpm and pH 7, followed by centrifugation to harvest cells. Using a Plasmid Purification Kit, pGEM-BPYNP was isolated from the harvested microbe.
  • pGEM-BPYNP was digested in 20 ul of a solution containing 10 U of NdeI and 10 U of HindIII, and analyzed by agarose electrophoresis as described above, and a 1.3 kbp BPYNP DNA fragment was purified using a gel extraction kit.
  • the two DNA fragments obtained as described above were incubated in a solution containing ligase of 3 U and 1X ligase buffer at 16°C for 18 hours.
  • JM109 E. coli cells were transformed with the reaction solution and cultured. From the resulting E. coli colonies, a plasmid was extracted.
  • the plasmid having the desired DNA fragment inserted therein was named “pFRPT-BPYNP”.
  • FIG. 3 schematically shows the plasmid.
  • the transformed strain obtained as described above was named “pFRPT-BPYNP/JM109”.
  • the strain was cultured using the medium composition shown in Table 4 below.
  • Table 4 Components of medium for culture of pFRPT-BPUNP/JM109
  • pFRPT-BPUNP/JM109 was inoculated into a 250 ml Erlenmeyer flask containing 25 ml of the medium shown in Table 4 above, and was shake-cultured overnight at 37°C and 240 rpm. 2 ml of the resulting culture was aseptically inoculated into the 200 ml medium of Table 4 in a 1-L Erlenmeyer flask, and was shake-cultured at 37°C and 240 rpm. When an absorbance of 0.8 was reached, IPTG (isopropyl-1-thio- ⁇ -D-galactopyranoside, Carbosynth) was added to a concentration of 1 mM.
  • IPTG isopropyl-1-thio- ⁇ -D-galactopyranoside, Carbosynth
  • the resulting culture was centrifuged at 8000 rpm for 10 minutes and washed with 20 ml of 10 mM phosphate buffer. Through this procedure, the source of purine nucleoside phosphorylase enzyme was obtained.
  • the strain was cultured using the medium shown in Table 4 above.
  • pFRPT-BPYNP/JM109 was inoculated into a 250 ml Erlenmeyer flask containing 25 ml of the medium shown in Table 4 above, and was shake-cultured overnight at 37°C and 240 rpm. 2 ml of the resulting culture was aseptically inoculated into the 200 ml medium of Table 4 in a 1-L Erlenmeyer flask, and was shake-cultured at 37°C and 240 rpm. When an absorbance of 0.8 was reached, IPTG (isopropyl-1-thio- ⁇ -D-galactopyranoside, Carbosynth) was added to a concentration of 1 mM.
  • IPTG isopropyl-1-thio- ⁇ -D-galactopyranoside, Carbosynth
  • the resulting culture was centrifuged at 8000 rpm for 10 minutes, and washed with 20 ml of 10 mM phosphate buffer. Through this procedure, the source of pyrimidine nucleoside phosphorylase enzyme was obtained.
  • 3'-amino-3'-deoxythymidine As a precursor for synthesis of 3'-amino-2',3'-dideoxyadenosine, 3'-amino-3'-deoxythymidine (ATMD) was synthesized.
  • ATMD 3'-amino-3'-deoxythymidine
  • 1 kg (3.74 mol) of 3'-azido-3'-deoxythymidine (AZT) was stirred together with 7.8 L of acetonitrile. 1.17 kg of triphenylphosphine (4.45 mol) was added thereto, followed by stirring at room temperature for 4 hours.
  • Each of a Bacillus stearothermophilus-derived purine nucleoside phosphorylase enzyme source (pFRPT-BPUNP/JM109) and a Bacillus stearothermophilus-derived pyrimidine nucleoside phosphorylase enzyme source (pFRPT-BPYNP/JM109) was suspended in purified water to a concentration of 20 U/ml, and then 1 ml of each of the suspension was added to the reaction solution .
  • coli-derived purine nucleoside phosphorylase enzyme source pFRPT-EPUNP/JM109; Korean Patent No. 0680765
  • thymidine phosphorylase pFRPT-TMDP/JM109; Korean Patent No. 0680765
  • reaction conversion rate of bioconversion to 3'-amino-2',3'-dideoxyadenosine (ADA) was calculated as follows:
  • ADA conversion rate (%) (concentration of produced ADA X 100) ⁇ (concentration of ATMD added to reaction)
  • Table 5 below shows the enzyme sources used for the reaction and the rate of conversion to 3'-amino-2',3'-dideoxyadenosine.
  • the use of the Bacillus stearothermophilus-derived nucleoside phosphorylases prepared according to the present invention showed the highest rate of conversion to 3'-amino-2',3'-dideoxyadenosine (ADA). Namely, it showed a higher rate of conversion to 3'-amino-2',3'-dideoxyadenosine (ADA) compared to the use of known E. coli-derived nucleoside phosphorylases or other phosphorylases.
  • the preferred amounts of pFRPT-BPUNP/JM109 and pFRPT-BPYNP/JM109 enzymes added to convert 83 mmol (1.85 M) of the substrate for 48 hours were determined to be 1120 U and 2240 U (13.5 U and 27.0 U per mmol of the substrate, which corresponds to a ratio of about 1:2), respectively.
  • each of the mixtures was shake-stirred at 40°C for 48 hours.
  • Each of the reaction solutions was analyzed by HPLC in the same manner as described in Example 5 above, and the rate of conversion to 3'-amino-2',3'-dideoxyadenosine was confirmed.
  • reaction substrates (3'-amino-3'-deoxythymidine and adenine) in a reaction solution for enzymatic conversion to 3'-amino-2',3'-dideoxyadenosine was set at 1:1, and substrates were added at varying concentrations of 0.1 M, 0.5 M, 1 M, 1.5 M and 2 M, and then suspended in 44.8 ml of 5 mM monobasic sodium phosphate solution.
  • adenine was added in varying amounts of 11.2 g (1.85 M, 1 equivalent) and 16.8 g (2.78 M, 1.5 equivalents). Then, each of the mixtures was suspended in 44.8 ml of 5 mM monobasic sodium phosphate solution, and 3.2 g (1120 U) of pFRPT-BPUNP/JM109 wet microbial cells and 3.2 g (2240 U) of pFRPT-BPYNP/JM109 wet microbial cells were added thereto, followed by shake-stirring at 40°C for 48 hours. Each of the reaction solutions was analyzed by HPLC in the same manner as described in Example 5 above, and the rate of conversion to 3'-amino-2',3'-dideoxyadenosine was confirmed.
  • the molar ratio of the substrates was set at 1:1 in order to minimize the production of reaction byproducts.
  • FIG. 4 shows HPLC chromatograms obtained in the individual purification processes.
  • the suspension was adjusted to a water content of 14-17%, and then stirred at 65°C for 1 hour and cooled slowly, after which it was cold-stirred at 4°C for 3 hours and filtered under reduced pressure to remove the remaining thymine.
  • the filtrate was completely concentrated, and was then suspended in 368 L of methyl alcohol. Then, the suspension was stirred at 65°C for 1 hour and cooled slowly, after which it was cold-stirred at 4°C for 3 hours and filtered.
  • 3'-amino-2',3'-dideoxyadenosine having an HPLC of 96.5% was recovered.
  • the recovered 3'-amino-2',3'-dideoxyadenosine was dissolved in 92 L of purified water and 460 L of methyl alcohol, and the solution was passed through a 0.2 ⁇ m filter and completely concentrated.
  • the concentrated residue was suspended in 368 L of methyl alcohol, and the suspension was stirred at 65 °C for 1 hour and cooled slowly, after which it was cold-stirred at 4°C for 3 hours, and filtered under reduced pressure, followed by drying in a vacuum at 55°C for 12 hours.
  • 58.02 kg (232 mol) of 3'-amino-2',3'-dideoxyadenosine having an HPLC purity of 99.5% was finally recovered.
  • the recovered 3'-amino-2',3'-dideoxyadenosine had a weight yield of 63.1% and a molar yield of 60.8%, based on the amount of 3'-amino-3'-deoxythymidine added.

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Abstract

La présente invention concerne un procédé de préparation de 3'-amino-2',3'-didésoxyadénosine à l'aide de nucléoside phosphorylases dérivées de Bacillus stearothermophilus. Selon le procédé de la présente invention, la 3'-amino-2',3'-didésoxyadénosine peut être produite en masse efficacement et économiquement par conversion enzymatique à haut débit et avec une pureté élevée de purification.
PCT/KR2017/003014 2016-03-21 2017-03-21 Procédé de préparation de 3'-amino-2',3'-didésoxyadénosine au moyen d'une nucléoside phosphorylase dérivée de bacillus Ceased WO2017164615A1 (fr)

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Cited By (1)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117143942A (zh) * 2023-10-30 2023-12-01 泰兴合全药业有限公司 3’-氨基-2’,3’-双脱氧鸟苷5’-三磷酸的合成方法

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