METHOD FOR ENZYMATIC PREPARATION OF FLUDARABINE PHOSPHATE

Abstract

A method for enzymatic preparation of fludarabine phosphate, comprising reaction of fludarabine with a high-energy phosphate compound under the action of deoxyribonucleic acid kinase. According to said method, acetate kinase and acetyl phosphate free acid or acetyl phosphate are also added. The technical problems present in the existing processes are successfully addressed by employing the enzymatic process to prepare the fludarabine phosphate. The usage of the high-energy phosphate compound is reduced by means of adding acetate kinase to recycle and regenerate a small amount of the high-energy phosphate compound, thereby reducing the generation of by-products having similar structures to the fludarabine phosphate, enhancing the operation convenience of purification steps in the industrial production of the fludarabine phosphate. The process is environment friendly, the reaction conditions are moderate, the cost is low, and the yield and the purity of the product obtained are high.

Claims

1. A method for enzymatic preparation of fludarabine phosphate, comprising a reaction of fludarabine with a high-energy phosphate compound under the action of a deoxyribonucleic acid kinase, wherein the method further comprises adding an acetate kinase and an acetyl phosphate free acid or an acetyl phosphate.

2. The method according to claim 1, characterized in that the method comprises the following steps: (1) Adding a high-energy phosphate compound and an acetyl phosphate free acid or an acetyl phosphate to a fludarabine-containing reactant solution; (2) Adding a deoxyribonucleic acid kinase and an acetate kinase to the solution obtained in step (1).

3. The method according to claim 1, wherein the molar ratio of the acetyl phosphate free acid or acetyl phosphate to the fludarabine is (1.0-3.0):1; preferably (1.5-2.5):1.

4. The method according to claim 1, wherein the high-energy phosphate compound is added in an amount of 0.001-0.003 mol.Math.L.sup.−1.

5. The method according to claim 1, wherein the acetyl phosphate is acetyl phosphate dilithium.

6. The method according to claim 1, wherein the high-energy phosphate compound is selected from the group consisting of the free acid of ATP, the free acid of ADP, the salt of ATP and the salt of ADP; preferably, the high-energy phosphate compound is ATP disodium salt.

7. The method according to claim 1, wherein the deoxyribonucleic acid kinase and the acetate kinase are added in the form of a thallus, a thallus crushing liquid, or an immobilized enzyme.

8. The method according to claim 7, wherein in the step (1), a soluble salt of Mg.sup.2+ is added to the fludarabine-containing reactant solution; preferably, the soluble salt of Mg.sup.2+ is MgCl.sub.2.6H.sub.2O or MgCl.sub.2.

9. The method according to claim 1, wherein during the reaction, the pH is adjusted to 6.0-9.0; preferably, the pH is adjusted to 7.5-8.5.

10. The method according to claim 1, wherein the reaction temperature is 30-45° C.; preferably, the reaction temperature is 37-40° C.

11. The method according to claim 2, wherein the molar ratio of the acetyl phosphate free acid or acetyl phosphate to the fludarabine is (1.0-3.0):1; preferably (1.5-2.5): 1.

12. The method according to claim 2, wherein the high-energy phosphate compound is added in an amount of 0.001-0.003 mol.Math.L.sup.−1.

13. The method according to claim 2, wherein the acetyl phosphate is acetyl phosphate dilithium.

14. The method according to claim 3, wherein the acetyl phosphate is acetyl phosphate dilithium.

15. The method according to claim 4, wherein the acetyl phosphate is acetyl phosphate dilithium.

16. The method according to claim 2, wherein the high-energy phosphate compound is selected from the group consisting of the free acid of ATP, the free acid of ADP, the salt of ATP and the salt of ADP; preferably, the high-energy phosphate compound is ATP disodium salt.

17. The method according to claim 3, wherein the high-energy phosphate compound is selected from the group consisting of the free acid of ATP, the free acid of ADP, the salt of ATP and the salt of ADP; preferably, the high-energy phosphate compound is ATP disodium salt.

18. The method according to claim 2, wherein the deoxyribonucleic acid kinase and the acetate kinase are added in the form of a thallus, a thallus crushing liquid, or an immobilized enzyme.

19. The method according to claim 2, wherein during the reaction, the pH is adjusted to 6.0-9.0; preferably, the pH is adjusted to 7.5-8.5.

20. The method according to claim 2, wherein the reaction temperature is 30-45° C.; preferably, the reaction temperature is 37-40° C.

Description

EMBODIMENTS

[0037] The technical solutions and effects of the present invention will be further described below through specific examples. The following examples are only used to illustrate the content of the present invention, and are not intended to limit the protection scope of the present invention. The simple modifications of the present invention applying the concepts thereof are all within the protection scope of the present invention.

[0038] In the following examples, all materials are commercially available unless otherwise specified.

[0039] Fludarabine was purchased from Shanghai Zhao Wei Technology Development Co., Ltd., lot No.: FARA17A1;

[0040] Expression vector pET24a and Escherichia coli E. coli BL21 (DE3) were purchased from Invitrogen company;

[0041] Vector ESR-2 was purchased from Tianjin Nankai Hecheng Science & Technology Co., Ltd.

[0042] The construction of recombinant Escherichia coli expressing the deoxyribonucleic acid kinase and the acetate kinase: the deoxyribonucleic acid kinase (dnk, NCBI-Gene ID: 42273) gene and the acetate kinase (ackA, NCBI-Gene ID: 8181271) gene were directionally cloned onto the expression vector pET24a. The segment between the NdeI site and the BamHI site of the expression vector pET24a was replaced by the deoxyribonucleic acid kinase gene, and the segment between the BamHI site and the EcoRI site of the expression vector pET24a was replaced by the acetate kinase gene to obtain recombinant expression plasmids. The recombinant expression plasmids were genetic sequenced and identified. The results were consistent with expectations. The recombinant expression plasmids were then transferred into Escherichia coli E. coli BL21(DE3). The construction methods of the expression vectors and recombinant Escherichia coli referred to example No. 3 and example No. 5 in patent WO 00/39307 respectively. After identification by enzyme digestion, recombinant Escherichia coli were successfully constructed.

[0043] Obtaining of Wet Thallus:

[0044] Mediums were prepared, 600 ml of LB medium with a formula of: peptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L, pH=7.2; 30 L of TB medium with a formula of: peptone 12 g/L, yeast extraction 24 g/L, glycerin 5 g/L, monopotassium phosphate 2.13 g/L, dipotassium hydrogen phosphate trihydrate 16.43 g/L, pH=7.0-7.5;

[0045] Fermentation culture conditions: recombinant Escherichia coli expressing the deoxyribonucleic acid kinase and acetate kinase was inoculated into the LB liquid medium, was cultured overnight at 37° C. and 200 rpm, and then inoculated into TB medium at an inoculation amount of 2%, was cultured until OD=6-8 at 37° C. and 200 rpm, and then kanamycin with a final concentration of 50 mg/L was added into the TB medium, the culture was induced overnight at 25° C.;

[0046] After 24h, OD=24 was measured, the fermentation was stopped, then centrifuged at 8000 rpm for 15 min, 1.2 kg of wet thallus was collected, frozen at −20° C., preserved and stand by.

[0047] Obtaining of Immobilized Enzyme:

[0048] The recombinant Escherichia coli expressing the deoxyribonucleic acid kinase and the acetate kinase was immobilized on the vector of ESR-2 with the bacterial load ratio of 2:1, and the immobilization method refers to SCI of 2.4 in A novel immobilization method for nuclease P.sub.1 on macroporous absorbent resin with glutaraldehyde cross-linking and determination of its properties, Process Biochemistry, 47(2012):665-670.

Example 1

[0049] Into a four-necked flask of 250 mL, 0.77 g of ammonium acetate, 0.08 g of MgCl.sub.2.6H.sub.2O, 13.3 g of acetyl phosphate dilithium (about 0.0875 mol), 0.36 g of ATP disodium salt (0.003 mol.Math.L.sup.−1) and 10 g of fludarabine (about 0.035 mol) were added, 100 ml of tap water was added to the reaction system, and stirred it well, and the pH was adjusted to 8.0 with 6 mol.Math.L.sup.−1 NaOH, the mixture was warmed up to 40° C. And then 10 g of wet thallus containing the acetate kinase and the deoxyribonucleic acid kinase was weighed, dissolved in 100 ml of water, then was added to the reaction system, the temperature was maintained at 37-40° C., the pH during the reaction was controlled at 7.5-8.5 with NaOH. The reaction was completed after 5 hours, the conversion rate was >99.0%, and the protein was removed, concentrated hydrochloric acid was used to adjust the pH to 2.0 at room temperature, a large amount of white crystals precipitated, then the system was cooled down to 5-10° C., maintained for 1 hour. 9.8 g of fludarabine phosphate product was obtained by filtration. The final product content was 98.3%, the HPLC purity was 99.0%.

Example 2

[0050] Into a four-necked flask of 250 mL, 0.77 g of ammonium acetate, 0.08 g of MgCl.sub.2.6H.sub.2O, 13.3 g of acetyl phosphate dilithium (about 0.0875 mol), 0.12 g of ATP disodium salt (0.001 mol.Math.L.sup.−1) and 10 g of fludarabine (about 0.035 mol) were added, 100 ml of tap water was added to the reaction system, and stirred it well, and the pH was adjusted to 8.0 with 6 mol.Math.L.sup.−1 NaOH, the mixture was warmed up to 40° C. And then 10 g of wet thallus containing the acetate kinase and the deoxyribonucleic acid kinase was weighed, dissolved in 100 ml of water, then was added to the reaction system, the temperature was maintained at 37-40° C., the pH during the reaction was controlled at 7.5-8.5 with NaOH. The reaction was completed after 7 hours, the conversion rate was 99.0%, and the protein was removed, concentrated hydrochloric acid was used to adjust the pH to 2.0 at room temperature, a large amount of white crystals precipitated, then the system was cooled down to 5-10° C., maintained for 1 hour. 9.7 g of fludarabine phosphate product was obtained by filtration. The final product content was 98.3%, the HPLC purity was 99.0%.

Example 3

[0051] Into a four-necked flask of 250 mL, 0.77 g of ammonium acetate, 0.08 g of MgCl.sub.2.6H.sub.2O, 7.98 g of acetyl phosphate dilithium (about 0.0525 mol), 0.36 g of ATP disodium salt (0.003 mol.Math.L.sup.−1) and 10 g of fludarabine (about 0.035 mol) were added. 100 ml of tap water was added to the reaction system, and stirred it well, and the pH was adjusted to 8.0 with 6 mol.Math.L.sup.−1 NaOH, the mixture was warmed up to 40° C. And then 10 g of wet thallus containing the acetate kinase and the deoxyribonucleic acid kinase was weighed, dissolved in 100 ml of water, then was added to the reaction system, the temperature was maintained at 37-40° C., the pH during the reaction was controlled at 7.5-8.5 with NaOH. The reaction was completed after 7 hours, the conversion rate was 99.0%, and the protein was removed, concentrated hydrochloric acid was used to adjust the pH to 2.0 at room temperature, a large amount of white crystals precipitated, then the system was cooled down to 5-10° C., maintained for 1 hour. 9.7 g of fludarabine phosphate product was obtained by filtration. The final product content was 98.5%, the HPLC purity was 99.1%.

Example 4

[0052] Into a four-necked flask of 250 mL, 0.77 g of ammonium acetate, 0.08 g of MgCl.sub.2.6H.sub.2O, 5.32 g of acetyl phosphate dilithium (about 0.035 mol), 0.36 g of ATP disodium salt (0.003 mol.Math.L.sup.−1) and 10 g of fludarabine (about 0.035 mol) were added. 100 ml of tap water was added to the reaction system, and stirred it well, and the pH was adjusted to 8.0 with 6 mol.Math.L.sup.−1 NaOH, the mixture was warmed up to 40° C. And then 10 g of wet thallus containing the acetate kinase and the deoxyribonucleic acid kinase was weighed, dissolved in 100 ml of water, then was added to the reaction system, the temperature was maintained at 37-40° C., the pH during the reaction was controlled at 7.5-8.5 with NaOH. The reaction was completed after 8 hours, the conversion rate was 90.2%, and the protein was removed, concentrated hydrochloric acid was used to adjust the pH to 2.0 at room temperature, a large amount of white crystals precipitated, then the system was cooled down to 5-10° C., maintained for 1 hour. 8.8 g of fludarabine phosphate product was obtained by filtration. The final product content was 96.0%, the HPLC purity was 97.6%.

Example 5

[0053] Into a four-necked flask of 250 mL, 0.77 g of ammonium acetate, 0.08 g of MgCl.sub.2.6H.sub.2O, 15.96 g of acetyl phosphate dilithium (about 0.105 mol), 0.36 g of ATP disodium salt (0.003 mol.Math.L.sup.−1) and 10 g of fludarabine (about 0.035 mol) were added. 100 ml of tap water was added to the reaction system, and stirred it well, and the pH was adjusted to 8.0 with 6 mol.Math.L.sup.−1 NaOH, the mixture was warmed up to 40° C. And then 10 g of wet thallus containing the acetate kinase and the deoxyribonucleic acid kinase was weighed, dissolved in 100 ml of water, then was added to the reaction system, the temperature was maintained at 37-40° C., the pH during the reaction was controlled at 7.5-8.5 with NaOH. The reaction was completed after 4 hours, the conversion rate was >99.0%, and the protein was removed, concentrated hydrochloric acid was used to adjust the pH to 2.0 at room temperature, a large amount of white crystals precipitated, then the system was cooled down to 5-10° C., maintained for 1 hour. 9.8 g of fludarabine phosphate product was obtained by filtration. The final product content was 96.2%, the HPLC purity was 99.0%.

Example 6

[0054] Into a four-necked flask of 250 mL, 0.77 g of ammonium acetate, 0.08 g of MgCl.sub.2.6H.sub.2O, 13.3 g of acetyl phosphate dilithium (about 0.0875 mol), 0.36 g of ATP disodium salt (0.003 mol.Math.LL.sup.−1) and 10 g of fludarabine (about 0.035 mol) were added. 100 ml of tap water was added to the reaction system, and stirred it well, and the pH was adjusted to 6.5 with 6 mol.Math.LL.sup.−1 NaOH, the mixture was warmed up to 40° C. And then 10 g of wet thallus containing the acetate kinase and the deoxyribonucleic acid kinase was weighed, dissolved in 100 ml of water, then was added to the reaction system, the temperature was maintained at 37-40° C., the pH during the reaction was controlled at 7.0-7.5 with NaOH. The reaction was completed after 7 hours, the conversion rate was 97.3%, and the protein was removed, concentrated hydrochloric acid was used to adjust the pH to 2.0 at room temperature, a large amount of white crystals precipitated, then the system was cooled down to 5-10° C., maintained for 1 hour. 9.6 g of fludarabine phosphate product was obtained by filtration. The final product content was 98.0%, the HPLC purity was 98.5%.

Example 7

[0055] Into a four-necked flask of 250 mL, 0.77 g of ammonium acetate, 0.08 g of MgCl.sub.2.6H.sub.2O, 13.3 g of acetyl phosphate dilithium (about 0.0875 mol), 0.36 g of ATP disodium salt (0.003 mol.Math.L.sup.−1) and 10 g of fludarabine (about 0.035 mol) were added. 100 ml of tap water was added to the reaction system, and stirred it well, and the pH was adjusted to 9.0 with 6 mol.Math.LL.sup.−1 NaOH, the mixture was warmed up to 40° C. And then 10 g of wet thallus containing the acetate kinase and the deoxyribonucleic acid kinase was weighed, dissolved in 100 ml of water, then was added to the reaction system, the temperature was maintained at 37-40° C., the pH during the reaction was controlled at 8.5-9.0 with NaOH. The reaction was completed after 5 hours, the conversion rate was >99.0%, and the protein was removed, concentrated hydrochloric acid was used to adjust the pH to 2.0 at room temperature, a large amount of white crystals precipitated, then the system was cooled down to 5-10° C., maintained for 1 hour. 9.5 g of fludarabine phosphate product was obtained by filtration. The final product content was 98.7%, the HPLC purity was 98.8%.

Example 8

[0056] Into a four-necked flask of 250 mL, 0.77 g of ammonium acetate, 0.08 g of MgCl.sub.2.6H.sub.2O, 13.3 g of acetyl phosphate dilithium (about 0.0875 mol), 0.36 g of ATP disodium salt (0.003 mol.Math.L.sup.−1) and 10 g of fludarabine (about 0.035 mol) were added. 100 ml of tap water was added to the reaction system, and stirred it well, and the pH was adjusted to 8.0 with 10 mol.Math.LL.sup.−1 NaOH, the mixture was warmed up to 30° C. And then 10 g of wet thallus containing the acetate kinase and the deoxyribonucleic acid kinase was weighed, dissolved in 100 ml of water, then was added to the reaction system, the temperature was maintained at 30-35° C., the pH during the reaction was controlled at 7.5-8.5 with NaOH. The reaction was completed after 7 hours, the conversion rate was 98.4%, and the protein was removed, concentrated hydrochloric acid was used to adjust the pH to 2.0 at room temperature, a large amount of white crystals precipitated, then the system was cooled down to 5-10° C., maintained for 1 hour. 9.64 g of fludarabine phosphate product was obtained by filtration. The final product content was 98.0%, the HPLC purity was 98.7%.

Example 9

[0057] Into a four-necked flask of 250 mL, 0.77 g of ammonium acetate, 0.08 g of MgCl.sub.2.6H.sub.2O, 13.3 g of acetyl phosphate dilithium (about 0.0875 mol), 0.36 g of ATP disodium salt (0.003 mol.Math.L.sup.−1) and 10 g of fludarabine (about 0.035 mol) were added. 100 ml of tap water was added to the reaction system, and stirred it well, and the pH was adjusted to 8.0 with 8 mol.Math.LL.sup.−1 NaOH, the mixture was warmed up to 45° C. And then 10 g of wet thallus containing the acetate kinase and the deoxyribonucleic acid kinase was weighed, dissolved in 100 ml of water, then was added to the reaction system, the temperature was maintained at 40-45° C., the pH during the reaction was controlled at 7.5-8.5 with NaOH. The reaction was completed after 4 hours, the conversion rate was >99.0%, and the protein was removed, concentrated hydrochloric acid was used to adjust the pH to 2.0 at room temperature, a large amount of white crystals precipitated, then the system was cooled down to 5-10° C., maintained for 1 hour. 9.55 g of fludarabine phosphate product was obtained by filtration. The final product content was 97.8%, the HPLC purity was 98.1%.

Example 10

[0058] Into a four-necked flask of 250 mL, 0.77 g of ammonium acetate, 0.08 g of MgCl.sub.2.6H.sub.2O, 13.3 g of acetyl phosphate dilithium (about 0.0875 mol), 0.28 g of ADP disodium salt (0.003 mol.Math.L.sup.−1) and 10 g of fludarabine (about 0.035 mol) were added. 100 ml of tap water was added to the reaction system, and stirred it well, and the pH was adjusted to 8.0 with 6 mol.Math.LL.sup.−1 NaOH, the mixture was warmed up to 40° C. And then 10 g of wet thallus containing the acetate kinase and the deoxyribonucleic acid kinase was weighed, dissolved in 100 ml of water, then was added to the reaction system, the temperature was maintained at 37-40° C., the pH during the reaction was controlled at 7.5-8.5 with NaOH. The reaction was completed after 5 hours, the conversion rate was >99.0%, and the protein was removed, concentrated hydrochloric acid was used to adjust the pH to 2.0 at room temperature, a large amount of white crystals precipitated, then the system was cooled down to 5-10° C., maintained for 1 hour. 9.76 g of fludarabine phosphate product was obtained by filtration. The final product content was 98.4%, the HPLC purity was 99.2%.

Example 11

[0059] Into a four-necked flask of 250 mL, 0.77 g of ammonium acetate, 0.08 g of MgCl.sub.2.6H.sub.2O, 15.2 g of acetyl phosphate diammonium salt (about 0.0875 mol), 0.36 g of ATP disodium salt (0.003 mol.Math.L.sup.−1) and 10 g of fludarabine (about 0.035 mol) were added. 100 ml of tap water was added to the reaction system, and stirred it well, and the pH was adjusted to 8.0 with 6 mol.Math.L.sup.−1 NaOH, the mixture was warmed up to 40° C. And then 10 g of wet thallus containing the acetate kinase and the deoxyribonucleic acid kinase was weighed, dissolved in 100 ml of water, then was added to the reaction system, the temperature was maintained at 37-40° C., the pH during the reaction was controlled at 7.5-8.5 with NaOH. The reaction was completed after 7 hours, the conversion rate was 98.0%, and the protein was removed, concentrated hydrochloric acid was used to adjust the pH to 2.0 at room temperature, a large amount of white crystals precipitated, then the system was cooled down to 5-10° C., maintained for 1 hour. 9.7 g of fludarabine phosphate product was obtained by filtration. The final product content was 98.1%, the HPLC purity was 99.0%.

Example 12

[0060] Into a four-necked flask of 250 mL, 0.77 g of ammonium acetate, 0.08 g of MgCl.sub.2.6H.sub.2O, 13.3 g of acetyl phosphate dilithium (about 0.0875 mol), 0.36 g of ATP disodium salt (0.003 mol.Math.L.sup.−1) and 10 g of fludarabine (about 0.035 mol) were added. 100 ml of tap water was added to the reaction system, and stirred it well, and the pH was adjusted to 8.0 with 6 mol.Math.L.sup.−1 NaOH, the mixture was warmed up to 40° C. And then 10 g of wet thallus containing the acetate kinase and the deoxyribonucleic acid kinase was weighed, dissolved in 100 ml of water, homogeneously breaked, then was added to the reaction system, the temperature was maintained at 37-40° C., the pH during the reaction was controlled at 7.5-8.5 with NaOH. The reaction was completed after 4 hours, the conversion rate was >99.0%, and the protein was removed, concentrated hydrochloric acid was used to adjust the pH to 2.0 at room temperature, a large amount of white crystals precipitated, then the system was cooled down to 5-10° C., maintained for 1 hour. 9.9 g of fludarabine phosphate product was obtained by filtration. The final product content was 98.7%, the HPLC purity was 99.6%.

Example 13

[0061] Into a four-necked flask of 250 mL, 0.77 g of ammonium acetate, 0.08 g of MgCl.sub.2.6H.sub.2O, 13.3 g of acetyl phosphate dilithium (about 0.0875 mol), 0.36 g of ATP disodium salt (0.003 mol.Math.L.sup.−1) and 10 g of fludarabine (about 0.035 mol) were added. 100 ml of tap water was added to the reaction system, and stirred it well, and the pH was adjusted to 8.0 with 6 mol.Math.L.sup.−1 NaOH, the mixture was warmed up to 40° C. And then 30 g of immobilized enzyme containing the acetate kinase and the deoxyribonucleic acid kinase was weighed and added to the reaction system, the temperature was maintained at 37-40° C., the pH during the reaction was controlled at 7.5-8.5 with NaOH. The reaction was completed after 8 hours, the conversion rate was 98.5%, and the protein was removed, concentrated hydrochloric acid was used to adjust the pH to 2.0 at room temperature, a large amount of white crystals precipitated, then the system was cooled down to 5-10° C., maintained for 1 hour. 9.1 g of fludarabine phosphate product was obtained by filtration. The final product content was 98.9%, the HPLC purity was 99.3%.