Methods for H-phosphonate synthesis of mono- and oligonucleotides
AssigneeHybridon, Inc.
InventorNandkumar Bhongle
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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).
Claims18
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.
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.
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.
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.
Dependencies are inferred from the claim text.