Methods for H-phosphonate synthesis of mono- and oligonucleotides

• US 5,705,629 A
• Filed: 10/20/1995
• Issued: 01/06/1998
• Est. Priority Date: 10/20/1995
• Status: Expired due to Fees

• Current Assignee: Nitto Denko Avecia, Inc. (Nitto Denko Corporation)
• Original Assignee: Hybridon, Inc. (Aceragen Incorporated)
• Inventors: Bhongle, Nandkumar
• Primary Examiner(s): Kunz, Gary L.
• Application Number:
  US08/546,318
• Time in Patent Office: 809 Days
• Field of Search: 536/25.34, 536/25.3
• US Class Current: 536/25.34
• CPC Class Codes: C07H 1/02 Phosphorylation

Abstract

New methods of synthesizing mono- and oligo- nucleotide H-phosphonates are disclosed. The methods comprise contacting a mononucleoside with phosphonic acid and benzoyl anyhydride to yield the corresponding mononucleoside H-phosphonate. Preferrably a catalytic amount of triphosgene is also used. A similar procedure can be used to couple a first mononucleoside to a second mononucleoside or to an oligonucleotide, the method comprising contacting, in the presence of benzoic anhydride and, preferrably, a catalytic amount of triphosgene, a mononucleotide or oligonucleotide having a free 5'"'"' hydroxyl with a mononucleoside having a 3'"'"' hydroxyl-bearing phosphorous moiety (preferably H-phosphonate).

Claims

1. A method of synthesizing a mononucleoside H-phosphonate comprising contacting a mononucleoside having a free 3'"'"' hydroxyl moiety with phosphonic acid and benzoic anhydride in a reaction mixture.

2. The method according to claim 1, further comprising adding a catalytic amount of triphosgene to the reaction mixture.

3. A method of synthesizing a dinucleotide comprising contacting a mononucleoside having hydroxyl-bearing phosphorous moiety at the 3'"'"' position with a mononucleoside having a free 5'"'"' hydroxyl group in the presence of benzoic anhydride in a reaction mixture.

4. The method according to claim 3, further comprising adding a catalytic amount of triphosgene to the reaction mixture.

5. The method according to claim 3 wherein the hydroxyl-bearing phosphorous moiety is H-phosphonate.

6. The method according to claim 4 wherein the hydroxyl-bearing phosphorous moiety is H-phosphonate.

7. A method of coupling a first mono- or oligo- nucleotide to a second mono- or oligo- nucleotide comprising contacting a first mono- or oligo- nucleotide having a hydroxyl-bearing phosphorous moiety at the 3'"'"' position with a second mono- or oligo- nucleotide having a free 5'"'"' hydroxyl group in the presence of benzoic anhydride in a reaction mixture.

8. The method according to claim 7, further comprising added a catalytic amount of triphosgene to the reaction mixture.

9. The method according to claim 7 wherein the hydroxyl-bearing phosphorous moiety is H-phosphonate.

10. The method according to claim 8 wherein the hydroxyl-bearing phosphorous moiety is H-phosphonate.

11. The method of claim 7 wherein the first and second nucleotides are mononucleotides.

12. The method of claim 8 wherein the first and second nucleotides are mononucleotides.

13. The method of claim 7 wherein the first nucleotide is a mononucleotide and the second nucleotide is a solid support-bound oligonucleotide.

14. The method of claim 8 wherein the first nucleotide is a mononucleotide and the second nucleotide is a solid support-bound oligonucleotide.

16. The method of claim 13 further comprising adding a catalytic amount of triphosgene to the reaction mixture in (a), (c), or both.

17. The method of claim 14 wherein the free 3'"'"' hydroxyl-bearing phosphorous moiety is H-phosphonate.

15. A method of synthesizing an oligonucleotide comprising sequentially:

(a) contacting a nascent mono- or oligo- nucleotide having a free 5'"'"' hydroxyl moiety with a 5'"'"' protected mono- or oligo- nucleotide having a free 3'"'"' H-phosphonate moiety in a reaction mixture containing benzoic anhydride to produce a nascent oligonucleotide bearing a 5'"'"' protecting group;

(b) cleaving the protecting group from the previously produced 5'"'"' protected nascent oligonucleotide to produce a nascent oligonucleotide having a free 5'"'"' hydroxyl moiety;

(c) optionally contacting the previously produced nascent oligonucleotide having a free 5'"'"' hydroxyl moiety with a 5'"'"' protected mono- or oligo- nucleotide having a free 3'"'"' H-phosphonate moiety to produce a 5'"'"' protected nascent oligonucleotide;

(d) optionally repeating (b) and (c) sequentially until an oligonucleotide of the desired sequence is obtained;

(e) oxidizing the oligonucleotide of (d) to yield a phosphodiester or phosphorothioate.

18. The method according to claim 15 wherein the nascent oligonucleotide is incrementally increased in length by one nucleotide at a time.