Method of synethesizing sulfurized oligonucleotide analogs
First Claim
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1. A method of synthesizing an oligonucleotide phosphorothioate or phosphorodithioate of a predetermined sequence, the method comprising the steps of:
- (a) providing a protected nucleoside or analog thereof attached to a solid phase support, the protected nucleoside or analog thereof having a blocked hydroxyl;
(b) deblocking the blocked hydroxyl to form a free hydroxyl;
(c) reacting with the free hydroxyl a blocked protected nucleoside phosphoramidite or phosphorthioamidite monomer or analog thereof to form a correct-sequence chain having a phosphorous(III) linkage and a blocked hydroxyl;
(d) sulfurizing the phosphorous(III) linkage by exposing the correct-sequence chain to a thiophosphorus compound selected from the group consisting of thiophosphoric acid polysulfide dithiophosphoric acid polysulfide, and dithiophosphinic acid polysulfide;
(e) repeating steps (b) through (d) until the oligonucleotide phosphorothioate or phosphorodithioate of the predetermined sequence is obtained.
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Abstract
A method for synthesizing sulfurized oligonucleotide analogs, such as phosphorothioate and phosphorodithioate analogs, is provided that employs a thiophosphorus compound, such as a thiophosphoric, dithiophosphoric, thiophosphinic, or dithiophosphinic acid disulfide or polysulfide, as a sulfurizing agent. The method of the invention may be used to sulfurize any phosphorous(III)-containing intermediate. Preferably, the method is practiced on a commercial DNA synthesizer using phosphoramidite and/or phosphorthioamidite intermediates.
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Citations
17 Claims
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1. A method of synthesizing an oligonucleotide phosphorothioate or phosphorodithioate of a predetermined sequence, the method comprising the steps of:
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(a) providing a protected nucleoside or analog thereof attached to a solid phase support, the protected nucleoside or analog thereof having a blocked hydroxyl; (b) deblocking the blocked hydroxyl to form a free hydroxyl; (c) reacting with the free hydroxyl a blocked protected nucleoside phosphoramidite or phosphorthioamidite monomer or analog thereof to form a correct-sequence chain having a phosphorous(III) linkage and a blocked hydroxyl; (d) sulfurizing the phosphorous(III) linkage by exposing the correct-sequence chain to a thiophosphorus compound selected from the group consisting of thiophosphoric acid polysulfide dithiophosphoric acid polysulfide, and dithiophosphinic acid polysulfide; (e) repeating steps (b) through (d) until the oligonucleotide phosphorothioate or phosphorodithioate of the predetermined sequence is obtained. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. In a method for synthesizing a sulfurized oligonucleotide, the method of the type wherein nucleosides are added stepwise to a growing oligonucleotide chain attached to a solid phase support and wherein phosphorous(III) linkages of the growing oligonucleotide chain are sulfurized, an improvement comprising:
sulfurizing the phosphorous(III) linkages of the growing oligonucleotide chain by exposing the growing oligonucleotide chain to a compound selected from the group consisting of thiophosphoric acid polysulfide, dithiophosphoric acid polysulfide, thiophosphinic acid polysulfide, and dithiophosphinic acid polysulfide. - View Dependent Claims (12, 13, 14, 15, 16, 17)
Specification