US5264566A
Expired / Lapsed
US
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Method for in vitro oligonucleotide synthesis using H-phosphonates
Assignee: Genentech Inc
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Filing: 1990-04-26
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Granted: 1993-11-23
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Expires: 2010-04-26
Abstract
Abstract A method is provided for the high fidelity, rapid and economical in vitro synthesis of oligonucleotides. Nucleoside H-phosphonates are condensed in seriatim using a dehydrating agent to produce a poly (nucleoside H-phosphonate). The product is oxidized to yield the desired oligonucleotide. A novel reagent is provided for multiple nucleoside additions in single cycles.
Inventors
- 👤 Brian C. Froehler
- 👤 Mark D. Matteucci
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 23:18
CPC Classifications
C; C07; C07H; C07H21/00
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
56 words
A method for preparing a polynucleotide phosphoramidate, said method comprising the steps of: oxidizing a carrier-bound polynucleoside H-phosphonate having more than two nucleosides with an oxidizing agent capable of oxidizing said carrier-bound polynucleoside H-phosphonate so as to yield carrier-bound polynucleoside phosphoramidate; and decoupling said polynucleoside phosphoramidate from said carrier so as to yield said polynucleotide phosphoramidate.
Claim 2
Independent Claim
56 words
A method for preparing a polynucleotide phosphoramidate, said method comprising the steps of: oxidizing a carrier-bound polynucleoside H-phosphonate having more than two nucleosides with an oxidizing agent capable of oxidizing said carrier-bound polynucleoside H-phosphonate so as to yield carrier-bound polynucleoside phosphoramidate; and decoupling said polynucleoside phosphoramidate from said carrier so as to yield said polynucleotide phosphoramidate.
Claim 3
Dependent Claim
20 words
A method as recited in claim 1 wherein a polymer carrier is bound to a 3'-hydroxyl of said polynucleoside H-phosphonate.
Claim 4
Dependent Claim
31 words
A method as recited in claim 1 wherein said polynucleoside H-phosphonate is bound to a carrier of material selected from the group consisting of controlled pore glass, silica gel and polystyrene.
Claim 5
Dependent Claim
26 words
A method as recited in claim 1 wherein said oxidizing agent is selected from the group consisting of iodine, N-chlorosuccinimide, N-bromosuccinimide, and salts of periodic acids.
Claim 6
Dependent Claim
69 words
A method as recited in claim 1 wherein said carrier-bound polynucleoside H-phosphonate has at least 40 nucleosides and wherein said oxidizing step occurs: with a relatively weak basic solution containing an amine with 1-5 S, N, or O atoms and fewer than 10 carbon atoms; and then with a relatively strong basic solution containing an amine with 1-5 S, N, or O atoms and fewer than 10 carbon atoms.
Claim 7
Dependent Claim
26 words
A method as recited in claim 1 wherein said decoupling step involves incubating a carrier to which said polynucleoside phosphoramidate is bound in concentrated ammonium hydroxide.
Claim 8
Dependent Claim
44 words
A method as recited in claim 1 wherein said oxidizing step occurs in a solution containing a base selected from the group comprising ammonia, primary amines and secondary amines, said base containing 1-5 S, N, or O atoms and fewer than 10 carbon atoms.
Claim 9
Dependent Claim
18 words
A method as recited in claim 7 wherein said amine includes an alkyl, an aryl, or an heteroaryl.
Claim 10
Dependent Claim
15 words
A method as recited in claim 7 wherein said amine is a heterocyclic secondary amine.
Claim 11
Dependent Claim
21 words
A method as recited in claim 7 wherein said solvent is selected from the group consisting of tetrahydrofuran and carbon tetrachloride.
Claim 12
Independent Claim
88 words
A method for preparing a polynucleotide phosphoramidate, said method comprising the steps of: oxidizing a carrier-bound polynucleoside H-phosphonate having more than two nucleosides in a solution of aqueous iodine and a base selected from the group consisting of NH 3 , MeNH 2 and n-BuNH 2 in a solvent selected from the group consisting of tetrahydrofuran and carbon tetrachloride so as to yield carrier-bound polynucleoside phosphoramidate; and incubating a carrier to which said polynucleoside phosphoramidate is bound in concentrated ammonium hydroxide so as to yield said polynucleotide phosphoramidate.
Claim 13
Independent Claim
88 words
A method for preparing a polynucleotide phosphoramidate, said method comprising the steps of: oxidizing a carrier-bound polynucleoside H-phosphonate having more than two nucleosides in a solution of aqueous iodine and a base selected from the group consisting of NH 3 , MeNH 2 and n-BuNH 2 in a solvent selected from the group consisting of tetrahydrofuran and carbon tetrachloride so as to yield carrier-bound polynucleoside phosphoramidate; and incubating a carrier to which said polynucleoside phosphoramidate is bound in concentrated ammonium hydroxide so as to yield said polynucleotide phosphoramidate.
References Cited (Backward Citations)
US4959463A
Cited
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| 3.5 Years (Year 4) | 1996-11-22 | 1997-05-24 | 1997-11-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2000-11-22 | 2001-05-24 | 2001-11-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2004-11-22 | 2005-05-24 | 2005-11-23 | $7700.00 | Window Closed / Lapsed |