US4507433A
Expired / Lapsed
US
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Preparation of oligodeoxyribonucleoside alkyl or arylphosphonates
Assignee: Johns Hopkins University
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Filing: 1983-10-07
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Granted: 1985-03-26
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Expires: 2003-10-07
Abstract
Abstract Process for synthesizing deoxyribonucleoside methylphosphonates on polystyrene polymer supports which involves condensing 5'-dimethoxytrityldeoxynucleoside 3'-methylphosphonates. The oligomers are removed from the support and the base protecting groups hydrolyzed by treatment with ethylenediamene in ethanol, which avoids hydrolysis of the methylphosphonate linkages. Two types of oligomers are synthesized: those containing only methylphosphonate linkages, d-Np(Np)nN, and those which terminate with a 5' nucleotide residue, dNp(Np)nN. The latter oligomers can be phosphorylated by polynucleotide kinase, and are separated by polyacrylamide gel electrophoresis according to their chain length. Piperidine randomly cleaves the oligomer methylphosphonate linkages and generates a series of shorter oligomers whose number corresponds to the length of the original oligomer. Apurinic sites introduced by acid treatment spontaneously hydrolyze to give oligomers which terminate with free 3' and 5'-OH groups. These reactions may be used to characterize the oligomers.
Inventors
- 👤 Paul S. Miller
- 👤 Paul O. P. Ts'o
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 16:09
CPC Classifications
C; C07; C07H; C07H21/00; Y; Y02; Y02P; Y02P20/00; Y02P20/50; Y02P20/55
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
111 words
A process for synthesizing oligonucleoside alkyl or arylphosphonates which comprises condensing (1) a 5'-protected nucleoside 3'-alkyl or arylphosphonate alkylammonium salt with (2) a 5'-hydroxy nucleoside or oligomer thereof attached through its 3' position to a polymer support whereby condensation takes place between the 5'-hydroxy group of (2) and the alkylammonium group of (1), repeating said condensation if and as appropriate to obtain the oligonucleoside alkyl or aryl phosphonate of desired length attached to the support and then cleaving the oligomer product from the support by treating the same with a diamino alkane whereby the oligomer is cleaved from the support and base protecting groups are removed without hydrolysis of phosphonate linkages.
Claim 2
Dependent Claim
111 words
A process for synthesizing oligonucleoside alkyl or arylphosphonates which comprises condensing (1) a 5'-protected nucleoside 3'-alkyl or arylphosphonate alkylammonium salt with (2) a 5'-hydroxy nucleoside or oligomer thereof attached through its 3' position to a polymer support whereby condensation takes place between the 5'-hydroxy group of (2) and the alkylammonium group of (1), repeating said condensation if and as appropriate to obtain the oligonucleoside alkyl or aryl phosphonate of desired length attached to the support and then cleaving the oligomer product from the support by treating the same with a diamino alkane whereby the oligomer is cleaved from the support and base protecting groups are removed without hydrolysis of phosphonate linkages.
Claim 3
Dependent Claim
22 words
The process of claim 1 wherein the polymer is polystyrene and the protected nucleoside 3'-alkyl or arylphosphonate is a protected nucleoside 3'-methylphosphonate.
Claim 4
Dependent Claim
10 words
The process of claim 2 wherein the polystyrene is crosslinked.
Claim 5
Dependent Claim
16 words
The process of claim 3 wherein the protected nucleoside is a protected nucleoside 3'-methylphosphonate triethylammonium salt.
Claim 6
Dependent Claim
15 words
The process of claim 1 wherein the diamino alkane is ethylene diamine dissolved in ethanol.
Claim 7
Dependent Claim
14 words
The process of claim 5 wherein the treatment is carried out at room temperature.
Claim 8
Dependent Claim
13 words
The process of claim 1 wherein the support is dried before the condensation.
Claim 9
Dependent Claim
19 words
The process of claim 7 wherein the drying of the support is carried out by coevaporation with anhydrous pyridine.
Claim 10
Dependent Claim
15 words
The process of claim 1 wherein the synthesized oligomer contains only alkyl or arylphosphonate linkages.
Claim 11
Dependent Claim
15 words
The process of claim 9 wherein the synthesized oligomer is purified by reversed phase chromatography.
Claim 12
Dependent Claim
15 words
The process of claim 1 wherein the synthesized oligomer is terminated with a 5'-nucleotide residue.
Claim 13
Dependent Claim
19 words
The process of claim 11 wherein the synthesized oligomer is purified by ion exchange chromatography and reversed phase chromatography.
Claim 14
Dependent Claim
20 words
The process of claim 11 wherein the oligomer is phosphorylated by polynucleotide kinase and is separated by polyacrylamide gel electrophoresis.
Claim 15
Dependent Claim
21 words
The process of claim 1 including treatment of the synthesized oligomer with piperidine so as to cleave the oligomer methylphosphonate linkages.
Claim 16
Dependent Claim
33 words
The process of claim 1 wherein the synthesized oligomer is treated with acid to form apurinic sites which are spontaneously hydrolyzed to give oligomers which terminate with free 3' and 5' OH groups.
References Cited (Backward Citations)
US4056673A
Cited
WO1982003079A1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1988-03-25 | 1988-09-24 | 1989-03-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-03-25 | 1992-09-24 | 1993-03-26 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-03-25 | 1996-09-24 | 1997-03-26 | $7700.00 | Window Closed / Lapsed |