Enzymatic synthesis of 4′-ethynyl nucleoside analogs
The present invention relates to an enzymatic synthesis of 4′-ethynyl-2′-deoxy nucleosides and analogs thereof, for example EFdA, that eliminates the use of protecting groups on the intermediates, improves the stereoselectivity of glycosylation and reduces the number of process steps needed to make said compounds. It also relates to the novel intermediates employed in the process.
1. A method for synthesizing a 4′-ethynyl 2′-deoxy nucleoside or an analog thereof comprising combining compound 6.5:
with purine nucleoside phosphorylase and a nucleobase or an analog thereof, in a buffered solution containing a manganese (II) salt,
and wherein 2X + is (a) two protons, (b) one proton and one monovalent cation, (c) two monovalent cations wherein each cation is the same or different, or (d) one divalent cation.
2. The method of claim 1 wherein the 4′-ethynyl 2′-deoxy nucleoside is
3. The method of claim 2 further comprising isolating
4. The method of claim 1 for synthesizing a 4′-ethynyl 2′-deoxy nucleoside or an analog thereof further comprising combining compound 6
and phosphopentomutase with the purine nucleoside phosphorylase and the nucleobase or the analog thereof in the buffered solution containing a manganese (II) salt.
5. The method of claim 4 further comprising removing inorganic phosphate byproduct from the reaction mixture.
6. The method of claim 5 comprising removing inorganic phosphate byproduct from the reaction mixture by (a) adding sucrose phosphorylase and sucrose to the reaction mixture or (b) adding calcium, magnesium or manganese to the reaction mixture.
7. The method of claim 5 further comprising isolating the 4′-ethynyl 2′-deoxy nucleoside or the analog thereof.
8. The method of claim 5 wherein the 4′-ethynyl 2′-deoxy nucleoside is
9. The method of claim 8 further comprising isolating
10. The method of claim 4 further comprising the step of synthesizing compound 6, wherein the synthesis comprises combining compound 5
with acetaldehyde and deoxyribose-phosphate aldolase in an aqueous solution to produce compound 6;
wherein 2X + is (a) two protons, (b) one proton and one monovalent cation, (c) two monovalent cations wherein each cation is the same or different, or (d) one divalent cation.
11. The method of claim 10 wherein the synthesis of compound 6 is performed in a sealed vessel.
12. The method of claim 10 further comprising the step of synthesizing compound 5, wherein the synthesis comprises (i) combining compound 4
with pantothenate kinase in a buffered solution,
in the presence of a bi-valent metal salt,
with ATP as a phosphate source wherein the ATP is regenerated in situ, or
(ii) combining compound 9
with galactose oxidase in a buffered solution,
in the presence of oxygen, catalase and either a peroxidase or a chemical oxidant,
wherein 2X + is (a) two protons, (b) one proton and one monovalent cation, (c) two monovalent cations wherein each said cation is the same or different, or (d) one divalent cation.
13. The method of claim 12 wherein the ATP is regenerated in situ employing (a) acetyl phosphate and acetate kinase, or (b) pyruvate oxidase, catalase and acetate kinase in the presence of pyruvate, phosphate and oxygen or (c) a combination thereof.
14. The method of claim 13 wherein (a) the pantothenate kinase is immobilized or (b) the pantothenate kinase and the acetate kinase are immobilized.
15. The method of claim 12 further comprising the step of synthesizing compound 4, wherein the synthesis comprises combining compound 3
with (a) galactose oxidase, copper, catalase and (b) peroxidase or an oxidant;
in the presence of oxygen, in a buffered solution to produce compound 4.
16. The method of claim 15 wherein the galactose oxidase is immobilized.
17. The method of claim 12 further comprising the step of synthesizing compound 9
comprising combining compound 3
with pantothenate kinase in a buffered solution,
in the presence of a bi-valent metal salt,
with ATP as a phosphate source wherein the ATP is regenerated in situ,
to produce compound 9,
wherein 2X + is (a) two protons, (b) one proton and one monovalent cation, (c) two monovalent cations wherein each said cation is the same or different, or (d) one divalent cation.
18. The method of claim 10 wherein the deoxyribose-phosphate aldolase comprises a polypeptide sequence having at least 85% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 14.
19. The method of claim 4 wherein the phosphopentomutase comprises a polypeptide sequence having at least 85% sequence identity to SEQ ID NO: 8.
20. The method of claim 1 wherein the nucleobase is 2-fluoroadenine.
21. A method for synthesizing a 4′-ethynyl 2′-deoxy nucleoside or an analog thereof comprising combining compound 5
acetaldehyde and a nucleobase or an analog thereof,
with deoxyribose-phosphate aldolase, phosphopentomutase and purine nucleoside phosphorylase,
in a buffered solution containing a manganese (II) salt,
wherein 2X + is (a) two protons, (b) one proton and one monovalent cation, (c) two monovalent cations wherein each cation is the same or different, or (d) one divalent cation.
22. The method of claim 21 further comprising removing inorganic phosphate byproduct from the reaction mixture.
23. The method of claim 22 comprising removing inorganic phosphate byproduct from the reaction mixture by (a) adding sucrose phosphorylase and sucrose to the reaction mixture or (b) adding calcium, magnesium, or manganese to the reaction mixture.
24. The method of claim 22 further comprising isolating the 4′-ethynyl 2′-deoxy nucleoside or analog thereof.
25. The method of claim 22 wherein the 4′-ethynyl 2′-deoxy nucleoside is
26. The method of claim 25 further comprising isolating
27. The method of claim 21 wherein the nucleobase is 2-fluoroadenine.