Process for the enzymatic synthesis of nucleosides

• US 5,075,225 A
• Filed: 04/06/1989
• Issued: 12/24/1991
• Est. Priority Date: 04/06/1989
• Status: Expired due to Fees

• Current Assignee: Texas A&M University System
• Original Assignee: Texas A&M University System
• Inventors: Hennen, William J., Wong, Chi-Huey
• Primary Examiner(s): Lilling, Herbert J.
• Application Number:
  US07/334,699
• Time in Patent Office: 992 Days
• Field of Search: 435/87, 435/88, 435/194, 435/89
• US Class Current: 435/87
• CPC Class Codes: C12P 19/38 Nucleosides

Abstract

A process for synthesizing nucleosides is disclosed. The process includes the reaction of an alkylated nucleoside (such as 7-methylguanosine or 7-methylinosine) with a heterocyclic base (such as adenine, 3-deazaadenine or 1,2,4-triazole-3-carboxamide) in the presence of a nucleoside-forming enzyme to form a nucleoside that includes a glycosyl component, donate by the alkylated nucleoside, bonded to the heterocyclic base.

Claims

1. An essentially irreversible process for synthesizing nucleosides comprising:

(a) reacting in an essentially irreversible manner, in solution and in the presence of a nucleoside-forming enzyme (i) a first nucleoside comprising a first modified heterocyclic base, that has been modified with a substituent selected from the group consisting of alkyl, alkylphenyl, hydroxy alkyl, and hydroxy alkylphenyl substituents, bonded to a glycosyl component with (ii) a second unmodified heterocyclic base, the first modified heterocyclic base, when dissociated from the glycosyl component of the first nucleoside, providing a relatively poor substrate for the nucleoside-forming enzyme as compared to the second unmodified heterocyclic base, for a time sufficient to enable the first nucleoside to donate the glycosyl component in an essentially irreversible manner for bonding with the second unmodified heterocyclic base;

(b) yielding a mixture that includes (i) a second nucleoside comprising the second unmodified heterocyclic base bonded to the glycosyl component and (ii) the first modified heterocyclic base that has been dissociated from the glycosyl component, and

(c) isolating the resulting second nucleoside from the mixture.

2. The process of claim 1 wherein the first nucleoside is selected from the group consisting of modified adenosine, guanosine, inosine, uridine, cytidine, thymidine, deoxyadenosine, deoxyguanosine, deoxyinosine, deoxythymidine, deoxyuridine, and deoxycytidine.

3. The process of claim 1 wherein the first nucleoside is a modified nucleoside-5-phosphate selected from the group consisting of modified adenosine-5-phosphate, guanosine-5-phosphate, inosine-5-phosphate, thymidine-5-phosphate, uridine-5-phosphate, cytidine-5-phosphate, deoxyadenosine-5-phosphate, deoxyguanosine-5-phosphate, deoxyinosine-5-phosphate, deoxythymidine-5-phosphate, deoxyuridine-5-phosphate, and deoxycytidine-5-phosphate.

4. The process of claim 1 wherein the nucleoside-forming enzyme is selected from the group consisting of purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, uridine phosphorylase, thymidine phosphorylase, nucleoside ribosyltransferase, nucleoside deoxyribosyltransferase, adenine phosphoribosyltransferase, hypoxanthine phosphoribosyltransferase, uracil phosphoribosyltransferase, guanosine phosphorylase, and 5-methylthioadenosine phosphorylase.

5. An essentially irreversible process for synthesizing nucleosides comprising:

(a) reacting in an essentially irreversible manner, in solution and in the presence of a nucleoside-forming enzyme (i) an alkylated nucleoside comprising an alkylated heterocyclic base, that has been modified with an alkyl substituent, bonded to a glycosyl component with (ii) a heterocyclic base for a time sufficient to enable the alkylated nucleoside to donate the glycosyl component in an essentially irreversible manner for bonding with the heterocyclic base to yield a mixture that includes a nucleoside comprising the heterocyclic base bonded to the glycosyl component; and

(b) isolating the resulting nucleoside from the mixture.

6. The process of claim 5 wherein the glycosyl component of the alkylated nucleoside is selected from the group consisting of a ribose, a ribose analog, a ribose derivative, a deoxyribose, a deoxyribose analog, and a deoxyribose derivative.

7. The process of claim 6 wherein the glycosyl component is selected from the group consisting of a ribose, a ribose-5-phosphate, a deoxyribose, and a deoxyribose-5-phosphate.

8. The process of claim 7 wherein the alkylated heterocyclic base is selected from the group consisting of an alkylated purine, an alkylated purine analog, an alkylated purine derivative, an alkylated pyrimidine, an alkylated pyrimidine analog, and an alkylated pyrimidine derivative.

9. The process of claim 8 wherein the alkylated nucleoside is selected from the group consisting of a methylguanosine and a methylinosine.

10. The process of claim 5 wherein the heterocyclic base is selected from the group consisting of a purine, a purine analog, a purine derivative, a pyrimidine, a pyrimidine analog, and a pyrimidine derivative.

11. The process of claim 10 wherein the heterocyclic base is selected from the group consisting of adenine, deazaadenine and 1,2,4-triazole-3-carboxamide.

12. The process of claim 5 wherein the pH of the solution is maintained between about 6.8 and about 8.0.

13. An essentially irreversible process for synthesizing nucleosides comprising:

(a) reacting in an essentially irreversible manner, in solution and in the presence of a nucleoside phosphorylase enzyme and an effective amount of a phosphate (i) a first nucleoside comprising a first modified heterocyclic base, that has been modified with a substituent selected from the group consisting of alkyl, alkylphenyl, hydroxy alkyl, and hydroxy alkylphenyl substituents, bonded to a glycosyl component with (ii) a second unmodified heterocyclic base, the first modified heterocyclic base, when dissociated from the glycosyl component of the first nucleoside, providing a relatively poor substrate for the nucleoside-forming enzyme as compared to the second unmodified heterocyclic base, for a time sufficient to enable the first nucleoside to donate the glycosyl component in an essentially irreversible manner, for bonding with the second unmodified heterocyclic base;

(b) yielding a mixture that includes (i) a second nucleoside comprising the second unmodified heterocyclic base bonded to the glycosyl component and (ii) the first modified heterocyclic base that has been dissociated from the glycosyl component; and

(c) isolating the resulting second nucleoside from the mixture.

14. The process of claim 13 wherein the glycosyl component is selected from the group consisting of a ribose, a ribose analog, a ribose derivative, a deoxyribose, a deoxyribose analog, and a deoxyribose derivative.

15. The process of claim 14 wherein the first nucleoside comprises an alkylated heterocyclic base that has been modified with an alkyl substituent, selected from the group consisting of an alkylated purine, an alkylated purine analog, an alkylated purine derivative, an alkylated pyrimidine, an alkylated pyrimidine analog, and an alkylated pyrimidine derivative, bonded to a glycosyl component, selected from the group consisting of a ribose, a ribose-5-phosphate, a deoxyribose, and a deoxyribose-5-phosphate, and wherein the second unmodified heterocyclic base is selected from the group consisting of a purine, a purine analog, a purine derivative, a pyrimidine, a pyrimidine analog, and a pyrimidine derivative.

16. The process of claim 15 wherein the nucleoside phosphorylase enzyme is selected from then group consisting of a purine nucleoside phosphorylase and a pyrimidine nucleoside phosphorylase.

17. A process for synthesizing nucleosides comprising:

(a) reacting in solution and in the presence of a nucleoside phosphorylase enzyme and an effective amount of a phosphate (i) a first nucleoside, selected from the group consisting of a methylguanosine and a methylinosine, with (ii) a heterocyclic base, selected from the group consisting of adenine, deazaadenine, and 1,2,4-triazole-3-carboxamide, for a time sufficient to enable the first nucleoside to donate a ribose component for bonding with the heterocyclic base to yield a mixture that includes a second nucleoside comprising the heterocyclic base bonded to the ribose component; and

(b) isolating the resulting second nucleoside from the mixture.

18. The process of claim 17 wherein the nucleoside phosphorylase enzyme is purine nucleoside phosphorylase.

19. The process of claim 18 wherein the first nucleoside is selected from the group consisting of a 7-methylguanosine and a 7-methylinosine.