US4816571A
Expired / Lapsed
US
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Chemical capping by phosphitylation during oligonucleotide synthesis
Assignee: Inex Pharmaceuticals Corp
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Filing: 1987-06-04
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Granted: 1989-03-28
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Expires: 2007-06-04
Abstract
Abstract A method is provided for capping failure sequences in oligonucleotide synthesis by phosphitylation. A phosphite monoester is reacted with the 5' or 3' hydroxyl of the failure sequence between successive condensation steps in a synthesis procedure to form a 5' or 3' phosphite diester with the failure sequence. The phosphite diester substituent is inert with respect to subsequent reaction steps in the synthesis of the desired oligonucleotide product.
Inventors
- 👤 William A. Andrus
- 👤 J. William Efcavitch
- 👤 Lincoln J. McBride
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 03:00
CPC Classifications
C; C07; C07H; C07H21/00; C07F; C07F9/00; C07F9/02; C07F9/06; C07F9/08; C07F9/141; C07F9/142
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
77 words
A method of capping failure sequences in solid phase oligonucleotide synthesis, the method comprising the step of condensing a capping agent with a hydroxyl of a failure sequence, the capping agent being defined by the formula: ##STR5## wherein R is straight-chain, branch, or cyclic lower alkyl containing from 1 to 6 carbon atoms, electron withdrawing substituted lower alkyl, lower alkyl-substituted or halo-substituted aryl, or a nitrogen-, oxygen-, or sulfur-containing heterocycle having from 5 to 8 carbon atoms.
Claim 2
Independent Claim
77 words
A method of capping failure sequences in solid phase oligonucleotide synthesis, the method comprising the step of condensing a capping agent with a hydroxyl of a failure sequence, the capping agent being defined by the formula: ##STR5## wherein R is straight-chain, branch, or cyclic lower alkyl containing from 1 to 6 carbon atoms, electron withdrawing substituted lower alkyl, lower alkyl-substituted or halo-substituted aryl, or a nitrogen-, oxygen-, or sulfur-containing heterocycle having from 5 to 8 carbon atoms.
Claim 3
Dependent Claim
27 words
The method of claim 1 wherein said step of condensing includes reacting said capping agent with said hydroxyl in the presence of a sterically hindered acid chloride.
Claim 4
Dependent Claim
53 words
The method of claim 2 wherein R is a straight-chain, branched, or cyclic alkyl containing from 1 to 6 carbon atoms, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, or a beta-electron-withdrawing substituted ethyl, and wherein said sterically hindered acid chloride is defined by the formula: ##STR6## wherein R' is tert-butyl, sec-butyl, cyclohexyl, adamantyl, norbornyl, or phenyl.
Claim 5
Dependent Claim
37 words
The method of claim 3 wherein said capping agent is a salt defined by the formula: ##STR7## wherein X + is selected from the group consisting of ammonium, lower alkylammonium, pyridinium, lutidinium, cyclohexylammonium, and metal salt cations.
Claim 6
Dependent Claim
22 words
The method of claim 4 wherein X + is selected from the group consisting of triethylammonium, tetrabutylammonium, diisopropylethylammonium, pyridinium, lutidinium, and cyclohexylammonium.
Claim 7
Dependent Claim
19 words
The method of claim 5 wherein X + is selected from the group consisting of triethylammonium, tetrabutylammonium, and diisopropylammonium.
Claim 8
Dependent Claim
25 words
The method of claim 6 wherein R is a straight-chained or branched alkyl of 1 to 4 carbon atoms, phenylethyl, beta-cyanoethyl, morpholino-piperidino, thiomorpholino, or beta-nitroethyl.
Claim 9
Dependent Claim
21 words
The method of claim 6 wherein said sterically hindered acid chloride is present in an equimolar amount as said capping agent.
Claim 10
Independent Claim
118 words
A method of synthesizing an oligonucleotide of a predetermined sequence on a solid support, the method comprising the steps of: (a) deprotecting a 5'-protected oligonucleotide attached to the solid support to form a deprotected oligonucleotide; (b) reacting a 5'-protected nucleotide monomer with the deprotected oligonucleotide to form either a 5'-protected oligonucleotide or a failure sequence, the failure sequence having a 5' hydroxyl; (c) capping the failure sequence by reacting a capping agent with the 5' hydroxyl of the failure sequence, the capping agent being defined by the formula: ##STR8## wherein R is lower alkyl, electron-withdrawing substituted lower alkyl, or lower alkyl- or halo-substituted aryl; and (d) repeating steps (a)-(c) until the oligonucleotide of the predetermined sequence is obtained.
Claim 11
Independent Claim
118 words
A method of synthesizing an oligonucleotide of a predetermined sequence on a solid support, the method comprising the steps of: (a) deprotecting a 5'-protected oligonucleotide attached to the solid support to form a deprotected oligonucleotide; (b) reacting a 5'-protected nucleotide monomer with the deprotected oligonucleotide to form either a 5'-protected oligonucleotide or a failure sequence, the failure sequence having a 5' hydroxyl; (c) capping the failure sequence by reacting a capping agent with the 5' hydroxyl of the failure sequence, the capping agent being defined by the formula: ##STR8## wherein R is lower alkyl, electron-withdrawing substituted lower alkyl, or lower alkyl- or halo-substituted aryl; and (d) repeating steps (a)-(c) until the oligonucleotide of the predetermined sequence is obtained.
Claim 12
Dependent Claim
76 words
The method of claim 8 wherein R of said capping agent is lower alkyl; beta-cyano-, beta-nitro-, beta-phenylsulphonyl-, or beta-phenylester-substituted ethyl; lower alkyl- or halo-substituted phenyl; lower alkyl- or halo-substituted benzyl; morpholino; thiomorpholino; or piperidino; and wherein said step of capping includes reacting said capping agent with said 5' hydroxyl of said failure sequence in the presence of a sterically hindered acid chloride of the formula: ##STR9## wherein R' is tert-butyl, sec-butyl, cyclohexyl, adamantyl, norbornyl, or phenyl.
Claim 13
Dependent Claim
12 words
The method of claim 9 wherein R' is tert-butyl, norbornyl, or adamantyl.
Claim 14
Dependent Claim
25 words
The method of claim 10 wherein said capping agent is a salt defined by the formula: ##STR10## wherein X + is triethylammonium, tetrabutylammonium, or diisopropylethylammonium.
Claim 15
Dependent Claim
19 words
The method of claim 6 wherein R is a straight-chained or branched alkyl of 1 to 4 carbon atoms.
Claim 16
Dependent Claim
12 words
The method of claim 6 wherein R is morpholino, piperidino, or thiomorpholino.
Claim 17
Dependent Claim
19 words
The method of claim 9 wherein R is a straight-chained or branched alkyl of 1 to 4 carbon atoms.
Claim 18
Dependent Claim
12 words
The method of claim 9 wherein R is morpholino, piperidino, or thiomorpholino.
References Cited (Backward Citations)
US4458066A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1992-03-27 | 1992-09-26 | 1993-03-28 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-03-27 | 1996-09-26 | 1997-03-28 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-03-27 | 2000-09-26 | 2001-03-28 | $7700.00 | Window Closed / Lapsed |