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    • 专利摘要:
    Method of obtaining nucleosides in a single step in the presence of a nucleoside enzyme 2'-deoxyribosyltransferase (NDT) type II thermostable from Chroococcidiopsis Thermalis PCC 7203 (CtNDT). The present invention relates to an enzymatic process for obtaining natural and non-natural nucleosides with therapeutic activity through the use of CtNDT in a transglycosylation reaction obtaining high yields. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2689244A1
    申请号:ES201730659
    申请日:2017-05-05
    公开日:2018-11-12
    发明作者:Jesús FERNANDEZ LUCAS；Daniel HORMIGO CISNERO；Vicente Javier CLEMENTE SUAREZ；Jon DEL ARCO ARRIETA；Rodrigo ÁLVAREZ SÁNCHEZ；María MARTÍNEZ GONZÁLEZ；Javier GALINDO PEREZ；Javier ACOSTA BUENO
    申请人:Europea De Madrid S L U, University of；UNIVERSIDAD EUROPEA DE MADRID SLU；
    IPC主号:
    专利说明:

    PROCEDURE FOR OBTAINING NUCLEOSIDS IN ONE STEP IN THE PRESENCE OF A 2´-DEOXIRRIBOSILTRANSFERASA TYPE II THERMOSTABLE CHROOCOCCIDIOPSIS THERMALIS PCC 7203 (CtNDT) ENZYME
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     5

    FIELD OF THE INVENTION
    The present invention falls within the field of biotechnology. More specifically, it refers to obtaining natural and unnatural nucleosides with therapeutic activity through biotechnological methods. 10

    BACKGROUND OF THE INVENTION

    Nucleic acids are molecules of vital importance for living beings as they are involved in different types of biochemical processes, such as storage and transfer of genetic information. Therefore, numerous nucleic acid derived compounds (NADs) have been used in different research areas for various purposes.

    In this sense, various NADs are commonly used in the treatment of different types of cancer, lymphoproliferative diseases, viral infections (such as hepatitis or HIV) and 20 some inflammatory diseases, such as Crohn's disease, as well as starting materials for synthesis. of antisense oligonucleotides.

    The synthesis of NADs has been traditionally carried out by chemical methods that involve numerous steps of protection-deprotection of functional groups and 25 involve the presence of mixtures of stereoisomers, hindering the process of purification of the products and decreasing the overall yield of the reaction.

    In addition, the necessary use of contaminating organic re-agents and solvents, makes these processes unsustainable which, from an industrial point of view, makes it difficult to scale them. Due to this, these types of compounds have high prices, preventing their clinical use in many cases and limiting their application in industries such as pharmaceutical or food. In recent decades, the partial or total replacement of these chemical methods by enzymatic methods has become a common practice in the industry (Boryski J. Curr Org Chem 2008, 12: 309-325; Fresco-Taboada A, from Mata I, 35 Arroyo M, Fernández-Lucas J. Appl Microbiol Biotechnol 2013, 97: 3773–3785; Fernández-
    Lucas, J. Appl Microbiol Biotechnol 2015, 99, 4615-4627; Rozzell, J. D. Bioorg Med Chem 1999, 7: 2253-2261).

    The enzymatic synthesis of NADs by whole cells or enzymes is an interesting alternative to the traditional chemical methods that offers many advantages, such as the reaction in a single container (one-pot), under mild conditions of pH and temperature, with a total stereo , regal and enantioselectivity and under environmentally friendly conditions. Such reactions can be carried out by a single enzyme with the aim of carrying out a specific reaction in a single step ("one-pot, one step"), or by two or more enzymes that act sequentially or in parallel ( “One-pot, x 10 steps”) to get the desired products.

    Nucleoside deoxyribosyltransferases (NDTs) (EC 2.4.2.6) catalyze the transglycosylation reaction between pyric and / or pyrimidine bases (Fresco-Taboada A, de la Mata I, Arroyo M, Fernández-Lucas J. Appl Microbiol Biotechnol 2013, 97: 3773–3785) using a 15 2'-deoxyribonucleoside that acts as a donor and a nitrogen base as an acceptor. The reaction proceeds through a covalent enzyme-substrate intermediate generated by the nucleophilic attack of a catalytic glutamic acid (Glu) residue on the anomeric carbon of 2′-deoxyribonucleoside. Subsequently, this complex is attacked by a second base generating a 2′-deoxyribonucleoside of the same configuration as the initial 20 (β-anomer). The reaction carried out is as follows:

    where B1 and B2 are purine or pyrimidine.

    Depending on the specificity in the substrate recognition, there are two types of NDTs, nucleoside 2′-deoxyribosyltransferases type I (PDT), specific for puric bases, and nucleoside 2′-deoxyribosyltransferases type II (NDT), which catalyze the transglycosylation reaction between purines and / or pyrimidines. Both types of NDTs, NDT and PDT, accept different natural and unnatural bases, but are highly specific on 2′-deoxyribose. Taking into account that the NDTs operate in a regal manner (glycosylation at 30 through the N1 of pyrimidines and N9 of purines) and stereoselective (only the
    β-anomer) offer great potential as a biocatalyst for the industrial synthesis of NADs.

    Although this nucleoside synthesis process would in principle be an efficient alternative to traditional chemical synthesis methods, there is a problem at the time of its implementation as biocatalysts in industrial processes, since not all types of pigic bases or Pyrimidines used as a substrate to generate nucleoside analogs are soluble in aqueous media.

    This limits the concentration of substrate with which it is possible to work and in many cases it makes difficult the scaling of the process. Various types of alternatives are used to solve these types of problems, such as the addition of organic co-solvents to the reaction medium, the increase or decrease of the pH of the medium or the increase of factors such as temperature or pressure. The problem with this type of strategy is that it is not easy to find enzymes that are stable under these conditions, so the search for 15 enzymes from microorganisms that grow in extreme conditions of pH, temperature, salinity or pressure constitutes an area of interest .

    Thermophilic organisms are a type of organisms whose optimum growth temperatures are above 45 ° C. They are mostly microorganisms belonging to the Bacteria and Archaea domains, and, according to their optimum growth temperature, they can be divided into moderate thermophiles (45-65ºC), extreme thermophiles (65-80ºC) and hyperthermophiles (> 80ºC) .

    The use of enzymes from thermophiles, thermozymes, in biocatalysis is of special interest since its natural heat resistance, which allows work at high temperatures, is frequently associated with a series of additional advantages such as:
     i) Reduction of the viscosity of the medium, increase of the solubility and diffusion coefficient of substrates and / or products; 30
     ii) Efficient thermozyme purification when expressed in a mesophilic system such as E. coli or Saccharomyces cerevisiae by differential denaturation techniques;
     iii) Lower risk of bioreactor contamination by environmental mesophilic species above 60 ° C; 35
    iv) Increased resistance of the biocatalyst to denaturing agents and organic solvents;
    v) Higher performance because the reaction by-products are minimized, allowing for easier product recovery.
     5
    DESCRIPTION OF THE INVENTION

    The present invention relates to a method of obtaining nucleosides comprising an enzymatic transglycosylation reaction in a single step, between a donor nucleoside and an acceptor nitrogen base in the presence of a thermostable 2 nucleoside 2'-deoxyribosyltransferase enzyme from Chroococcidiopsis thermalis PCC 7203 (CtNDT) characterized by the sequence SEQ ID NO: 1.

    Chroococcidiopsis thermalis PCC 7203 is a strain of C. thermalis
     fifteen
    The nucleoside enzyme 2'-deoxyribosyltransferase can be obtained recombinantly by expressing the ndt gene of Chroococcidiopsis thermalis PCC 7203, characterized by SEQ ID NO: 2, in an accessible bacterium such as, for example, Escherichia coli. The recombinant enzyme can be obtained from sequences whose expression gives rise to polypeptides that retain 2'-deoxyribosyltransferase nucleoside activity. twenty

    Thanks to the use of CtNDT in the enzymatic transglycosylation reaction in a single step, different natural and unnatural nucleosides are obtained and, within the latter, arabinosyl-nucleosides such as adenine arabinoside (ara-A), cytosine (ara-C ), hypoxanthine (ara-H) and guanine (ara-G), 2 ′, 3′-dideoxyribosyl nucleosides such as didanosine (ddIno), as well as different 2'-fluoro-2'- deoxyribonucleosides such as 2'-fluoro -2'-deoxyadenosine (2'F-dAdo) and 2'-fluoro-2'-deoxyguanosine (2'F-dGuo). These natural nucleosides have therapeutic interest.

    In another aspect of the invention, the enzymatic transglycosylation reaction in a single step 30 is carried out in the presence of organic solvents in a concentration of 20% -40% by volume. It has been shown that both the activity of the enzyme and its stability are not affected by the presence of organic solvents in different concentrations within the range of 20% -40% by volume.
     35
    The organic solvents used for said reaction may be aprotic polar, alcohols and polyols. In a preferred embodiment the organic solvent is acetone, ethyl acetate, acetonitrile, chloroform, dimethylformamide, dimethyl sulfoxide, ethanol, isopropanol, methanol, glycerol, ethylene glycol or propylene glycol.
     5
    In another aspect of the invention, transglycosylation reactions are performed at different temperatures (20-90 ° C) and different pH values (3-11), preferably at a temperature between 40 ° C and 90 ° C and at a pH between 3 and 7 , since a retained activity value of more than 70% is observed.
     10
    In another aspect of the invention, the acceptor nitrogen base is adenine, guanine, hypoxanthine or cytosine.

    In another aspect of the invention, the donor nucleoside is 2′, 3′-dideoxyribosylnucleoside, 2′-fluoro-2′-deoxyribosylnucleoside, arabinosyladenine, arabinosylcytosine, arabinosylhypoxanthin, 15 2'-deoxycytidine, 2'-deoxyuridine, 2'-deoxyuridine, 2'-deoxyuridine , 2'-deoxyguanosine, 2'-deoxyinosine or 2'-deoxythymidine.

    FREE TEXT OF THE SEQUENCE LISTING
    Below is a translation of the free text in English that appears in the list of 20 sequences.

    SEQ ID NO: 1. Protein with nucleoside activity 2'-deoxyribosyltransferase type II of strain PCC 7203 of Chroococcidiopsis thermalis.
    SEQ ID NO: 2. Gene with nucleoside activity 2'-deoxyribosyltransferase type II of strain 25 PCC 7203 of Chroococcidiopsis thermalis.
    SEQ ID NO: 3. T7 Promoter Primer
    SEQ ID NO: 4. T7 Terminator Primer

     30
    BRIEF DESCRIPTION OF THE FIGURES
    Figure 1. Effect of temperature on CtNDT activity.
    Figure 2. Effect of pH on CtNDT activity.
    Figure 3. Effect of temperature on the stability of CtNDT by studies of thermal denaturation of the enzyme at various pH values in various buffers 35 (Sodium citrate pH 5-6 and Potassium phosphate, 6-7) at 60 ° C.
    Figure 4. Activity of CtNDT in the presence of 20% polar aprotic solvents (acetone, ethyl acetate, acetonitrile, chloroform, dimethylformamide, dimethyl sulfoxide), alcohols (ethanol, isopropanol, methanol) and polyols (glycerol, ethylene glycol and propylene glycol)

    DESCRIPTION OF EMBODIMENTS 5
    Having described the present invention, it is further illustrated by the following examples.

    Example 1. Construction of the recombinant microorganism E. coli CtNDT
    Based on the genome of the extremophilic cocoa cyanobacterium Chroococcidiopsis thermalis 10 PCC 7203 housed in the database of genetic sequences of the NIH (National Institutes of Health of the United States, GenBank), the bioinformatic search has been carried out in the same sequence of DNA that could encode possible putative 2'-deoxyribosyltransferases using the BlastN and BlastP algorithms respectively (Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJJ Mol. 15 Biol. 1990, 215: 403- 410) through the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/BLAST/) server.

    As a result, the gene sequence with GenBank accession number was found: AFY86715.1, which was named as ndtCt (SEQ ID No. 2). By multiple alignment of the amino acid sequence corresponding to the translation of the ndtCt gene, with sequences from other NDTs, it was observed that said gene could encode a 2′-deoxyribosyltransferase (CtNDT). To carry out this alignment, the Clustal  program was used (Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, Lopez R. Nucleic acids research 2010, 38 Suppl: W695-9) hosted on the server EMBL-EBI 25 (https://www.ebi.ac.uk/Tools/msa/clustalo/).

    Once the bioinformatic analysis of the gene of interest was carried out, the GenScript commercial house (http://www.genscript.com/) was commissioned to synthesize it, as well as its inclusion as an NdeI-EcoRI fragment within the expression vector pET28b (+) (Novagen) resulting in the recombinant plasmid pET28bCtNDT. Said vector carries a Kanamycin resistance marker, an IPTG inducible lac expression promoter for selection in this genus and a DNA sequence that would encode the presence of 6 histidines at the N-terminal end of the protein, resulting in His-CtNDT fusion protein. Then, the transformation was carried out by shock 35
    collision of competent E. coli BL21 (D3) cells previously obtained according to the RbCl procedure (Sambrook et al. (1989) Molecular Cloning: A laboratory Manual, Cold Spring Harbor, New York, USA) and recombinant clones expressing constitutively the ndtCt gene (named E. coli CtNDT). The transformants were selected in solid Luria-Bertani medium to which kanamycin had been added (50 5 µg / ml). Through this procedure several clones were obtained that were analyzed by electrophoresis of the purified plasmids, by the High Pure Plasmid Isolation Kit (Roche) procedure, in 1% agarose gels and subsequent sequencing using the T7 promoter oligonucleotides (SEQ IDNO: 3) and T7 reverse 5'- (SEQ ID NO: 4).
     10
    Example 2. Production and purification of CtNDT
    For the production of the CtNDT using the previously obtained recombinant clone, an isolated colony of the recombinant organism was grown in Luria-Bertani, LB medium (Sambrook et al. (1989) Molecular Cloning: A laboratory Manual, Cold Spring Harbor, New York, USA)), at 37 ° C and 250 rpm, in the presence of a kanamycin concentration of 50 15 g / ml until an optical density of 0.6-0.8 is obtained. At that time, IPTG was added to a final concentration of 0.5 mM leaving the culture growing for 3 hours under the conditions described above. After this time, it was centrifuged at 3500 x g for 30 minutes at 4 ° C, and the cell disruption of the sediment obtained was performed, by sonication in 10 mM sodium phosphate buffer pH = 7. After the process was finished, the lysed cells were centrifuged at 9500 x g 30 min at 4 ° C and the supernatant was separated from the cell debris. Said supernatant contains the His-CtNDT fusion protein, which allowed a simple purification by affinity chromatography. For this, the supernatant previously obtained was applied to a 5-ml HisTrap FF Ni-NTA column (GE Healthcare) previously equilibrated in buffer 1 (20 mM Tris-HCl buffer, 25 pH 8.0, 100 mM NaCl and 10 mM imidazole), and subsequently eluted in buffer 2 (20 mM Tris-HCl buffer, pH 8.0, 100 mM NaCl and 500 mM imidazole).

    Subsequently with the purest fractions a molecular exclusion chromatography was carried out by applying the sample on a Hi-Load 16/60 Superdex 30 200 prep grade column (GE Healthcare) column previously equilibrated with buffer 3 (20 mM Tris -HCl, pH 8.0). For the purification processes, an FPLC model Akta Prime (GE Healthcare) was used. The fractions were analyzed by protein electrophoresis under denaturing conditions in polyacrylamide gels in the presence of 0.1% SDS (SDS-PAGE). 35

    Example 3. Effect of pH, temperature and solvents on the activity and stability of CtNDT.
    For this, the enzyme activity was determined at different pH (4.5-11) and temperature (20-90 ° C) values, observing that the enzyme showed retention activity values greater than 70% over a wide range of temperature (40-90 ° C) (Figure 1) and pH (4-7) (Figure 2). Particularly interesting is the fact that the enzyme has activity values greater than 95% in the range 60-90 ° C, which would allow the processes to be carried out at high temperatures, with the consequent advantages that this would entail. Based on the results, the optimal conditions of use of the biocatalyst would be in the temperature range (60-90 ° C) and pH (4-6). As standard conditions for the quantification of the enzymatic activity, pH 6 and 60 ° C were chosen. Under these conditions, the biocatalyst showed an activity of 37 international activity unit (IU). In addition, the effect of temperature on the stability of the enzyme has been evaluated by studies of thermal denaturation of the enzyme at various pH values in various buffers 15 (Sodium citrate pH 5-6 and Potassium phosphate, 6-7) at 60 ºC (Figure 3).

    Additionally, the effect of the addition of organic solvents on the activity of the enzyme has been evaluated (Figure 4). In this sense, the activity of the enzyme has been evaluated in the presence of 20% organic solvents, such as aprotic polar solvents (acetone, ethyl acetate, acetonitrile, chloroform, dimethylformamide, dimethyl sulfoxide), alcohols (ethanol, isopropanol, methanol) and polyols (glycerol, ethylene glycol and propylene glycol). As can be seen in Figure 4, retained activity values greater than 60% percent were obtained in all cases. This tolerance of the enzyme to the use of organic solvents, together with the great thermostability shown by it, allows us to be optimistic about the possible process implementation at an industrial level.

    Example 4. Determination and characterization of deoxyribosyltransferase activity in CtNDT
    For the determination of protein activity, the standard activity test 30 was carried out, which consisted of the synthesis of 2'-deoxyadenosine from 2'-deoxyinosine and adenine. To that end, various enzyme concentrations were added to a solution of 10 mM 2'-deoxyinosine (dIno) and 10 mM Adenine (Ade) 50 mM phosphate buffer, pH = 6.0. The reaction was kept under stirring at 60 ° C and 350 rpm. in an interval between 0 and 30 minutes hours, in order to determine the time interval in which the enzyme 35
    Show a linear response. The reaction was stopped by adding a volume of methanol previously cooled to 4 ° C, and subsequently heated to 95 ° C in order to denature the enzyme. Subsequently after centrifugation, the products obtained were determined by High Performance Liquid Chromatography (HPLC) according to the methodology described (Fernández-Lucas J. et al. (2007) Enzyme Microb. 5 Technol. 40, 1147-1155). Under these reaction conditions, an international unit of activity (IU) is defined as the amount of biocatalyst that produces 1 μmol of 2′-deoxyadenosine per minute.

    Example 5. Synthesis of different nucleosides using CtNDT 10
    Next, the procedure for the synthesis of different nucleosides is explained by using as a biocatalyst of the CtNDT. In the different examples presented, the reaction products can be quantified by HPLC using the following conditions: ACE 5 C18-PFP column ( 250 x 4.6 mm); mobile phase: (1) linear gradient for 10 minutes of 0.1 M trimethylammonium acetate to reach 90/10 (v / v) 15 0.1 M trimethylammonium acetate / acetonitrile, (2) 10 minutes with 90/10 (v / v) 0.1 M trimethylammonium acetate / acetonitrile. The flow rate is set at 1 ml / min (180 bar pressure) and the UV detector is set at 260 nm. Under these conditions, retention times of detected bases and nucleosides are presented in Table I.
     twenty
    After determining the optimal conditions of CtNDT activity, substrate specificity studies were carried out, using different 2′-deoxynucleosides and bases, in order to determine which substrates the enzyme best recognizes. For this, the enzyme was incubated under optimal conditions of activity and stability described in the previous section using different 2'-deoxynucleosides (dAdo, dCyd, dGuo, dIno, dThd and dUrd) 10 10 mM and bases (Ade, Cyt, Hyp, Gua , Ura and Thy), The reaction conditions were:
     36 units of enzyme in 40 μl at 60 ºC, 5-10 min, 300 rpm. [Substrates] = 1 mM, 50 mM MES buffer, pH 6.5, for Ade, Cyt, Hyp, Ura and Thy, and
     0.6 μg of enzyme in 40 μl at 60 ºC, 5-10 min, 300 rpm. [Substrates] = 1 mM, 50 mM sodium borate buffer, pH 8.5, for Gua. 30

    As can be seen in Table II, CtNDT similarly recognizes as a donor 2'-deoxyadenosine (dAdo), 2′-deoxyinosin (dIno) and 2′-deoxyguanosine (dGuo), the same thing not happening with pyrimidine nucleosides, a exception of 2′-deoxycytidine. This is consistent with the results obtained by using various bases as acceptor, 35
    since, as can be seen, the enzyme recognizes as better substrates Adenine (Ade) and Hypoxanthine (Hyp), showing less activity on Cytosine (Cit) and zero on Uracil (Ura) and Thymine (Thy).

    Table I. Base retention times, 2’-deoxynucleosides and nucleosides 5 obtained.

    Table II CtNDT activity
    nd = not determined

    5.1. Synthesis of arabinosyl nucleosides through the use of CtNDT.
    For the synthesis of arabinosyl nucleosides, the procedure was as follows:
     225 units of enzyme in 30 deL of a solution containing arabinosyl nucleoside (ara-A, ara-C, ara-H or ara-G) 1 mM, [Base] = 1 mM, in 50 mM MES buffer at pH 6.5, for Ade, Hyp and Cyt.
     225 units of enzyme in 30 deL of a solution containing arabinosyl nucleoside (ara-A, ara-C, ara-H or ara-G), [Base] = 1 mM, in 50 mM sodium borate buffer at pH 8.5, for Gua.
    The reaction is kept under stirring at 60 ° C and 300 r.p.m. for a period of 24 hours. The corresponding yields are shown in table III. Particularly interesting is the obtaining of ara-A (Vidarabine), used for the treatment of the herpes virus and varicella-zoster viruses, in addition to being a potential precursor of antitumor drugs 15 such as fludarabine or clofarabine. On the other hand, ara-G, used to treat acute lymphoblastic T-cell leukemia (LAA-T) and lymphoblastic T-cell lymphoma (LLB-T).

    Table III Yields of arabinosyl nucleoside production
     Performance (%)
            Donor Acceptor  Ade 4 h 24 h Gua 4 h 24 h Hyp 4 h 24 h Cyt 4 h 24 h
     for A  - - 7 15 1 4.1 3 1
     ara G  7 14 - - n.d. 4 n.d. n.d
     ara H  2 9 2 11 - - n.d. n.d.
    nd = not determined

    5.2. Synthesis of 2′-fluoro-2′-deoxyribosylnucleosides through the use of CtNDT. 5
    For the synthesis of 2′-fluoro-2′-deoxyribosylnucleosides, we proceeded as follows:
     225 units of enzyme in 30 deL of a solution containing 2′-fluoro-2′-deoxyribosylnucleoside (2′F-dIno) 1 mM, [Base] = 1 mM, in 50 mM MES buffer at pH 6.5, for Ade, Hyp and Cyt.
     225 units of enzyme in 30 deL of a solution containing 2′-fluoro-2′-10 deoxyribosylnucleoside (2′F-dIno) 1 mM, [Base] = 1 mM, in 50 mM sodium borate buffer at pH 8 , 5, for Gua.
    The reaction is kept under stirring at 60 ° C and 300 rpm. for a period of 24 hours. The corresponding yields are shown in table IV. In this case we highlight the obtaining of 2´-fluoro-2´-deoxyguanosine (2´F-dGuo), used as antiviral 15 for the treatment of influenza virus.
    Table IV Production yields of 2′-fluoro-2′-deoxyribosylnucleosides
     Performance (%)
            Donor Acceptor  Ade 4 h 24 h Gua 4 h 24 h Hyp 4 h 24 h Cyt 4 h 24 h
     2′F-dIno  4 11 3 11 - - n.d. n.d.
    nd = not determined

     twenty
    5.3. Synthesis of 2 ′, 3′-dideoxyiribosyl nucleosides by using CtNDT.
    For the synthesis of 2 ′, 3′-dideoxyiribosyl nucleosides (Figure 3), the procedure was as follows:
     225 units of enzyme in 30 deL of a solution containing 2 ′, 3′-dideoxyribosyl nucleoside (ddAdo, ddIno), [Base] = 1 mM in 50 mM MES buffer at pH 6.5, for 5 Ade, Hyp and Cyt .
     225 units of enzyme in 30 deL of a solution containing 2 ′, 3′-dideoxyribosyl nucleoside (ddAdo, ddIno), [Base] = 1 mM in 50 mM sodium borate buffer at pH 8.5, for Gua.
    The reaction is kept under stirring at 60 ° C and 300 r.p.m. for a period of 24 hours. 10 The yields of the corresponding ones are shown in Table V. In this case we highlight the obtaining of didanosine (ddIno) a known antiviral for the treatment of HIV-1 infection.
    Table V. Production yields of 2 ′, 3′-dideoxyribosyl nucleosides
     Performance (%)
            Donor Acceptor  Ade 4 h 24 h Gua 4 h 24 h Hyp 4 h 24 h Cyt 4 h 24 h
     ddAdo  - - 6 n.d. n.d. 1 n.d. 5
     ddIno  12 1 3 n.d. - - n.d. n.d.
    nd = not determined 15

    权利要求:
    Claims (6)
    [1]

    1. A method of obtaining nucleosides comprising an enzymatic transglycosylation reaction in a single step, between a donor nucleoside and an acceptor nitrogenous base in the presence of the nucleoside 2'-deoxyribosyltransferase enzyme type II from Chroococcidiopsis thermalis PCC 7203 comprising the sequence SEQ ID NO: 1.

    [2]
    2. Method according to claim 1, characterized in that it is carried out in the presence of organic solvents in a concentration of 20% -40% by volume. 10

    [3]
    3. Method according to claim 2, characterized in that the organic solvent is selected from acetone, ethyl acetate, acetonitrile, chloroform, dimethylformamide, dimethyl sulfoxide, ethanol, isopropanol, methanol, glycerol, ethylene glycol or propylene glycol. fifteen

    [4]
    4. Method according to any of claims 1-3, characterized in that the acceptor nitrogen base is selected from adenine, guanine, hypoxanthine or cytosine.
     twenty
    [5]
    5. Method according to any of claims 1-4, characterized in that the donor nucleoside is selected from 2 ′, 3′-dideoxyribosylnucleoside, 2′-fluoro-2′-deoxyribosylnucleoside, arabinosyladenine, arabinosylcytosine, arabinosyl hypoxanthine, 2'-deoxycytidine , 2'-deoxyuridine, 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyinosin or 2'-deoxythymidine. 25

    [6]
    Method according to any one of claims 1-5, characterized in that it is carried out at a temperature between 40 ° C and 90 ° C and at a pH between 3 and 7.
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    同族专利:
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    ES2689244B2|2019-04-09|
    引用文献:
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