US4401796A
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US
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Solid-phase synthesis of polynucleotides
Assignee: EDWARDS S IRONS PC
•
Filing: 1981-04-30
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Granted: 1983-08-30
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Expires: 2001-04-30
Abstract
Abstract A resin support formed from a suitable resin such as polystyrene or a silica gel may be provided by forming an amino resin and by reacting the amino resin with an activated ester of a nucleoside to obtain the dimethoxytrityl resin. To add each successive nucleotide to the resin support, the nucleoside is phosphorylated to provide a monotriazolide. The phosphorylation may be provided by the reaction of a phosphoditriazolide with a 3'-hydroxyl neucleoside. The dimethoxytrityl resin is then hydroxylated to substitute the hydroxyl group for the dimethoxytrityl group in the resin support. The monotriazolide is then reacted with the hydroxylated resin support to provide the dimethoxytrityl resin. This reaction may occur in the presence of a nucleophilic catalyst, such as a catalyst selected from a group consisting of dimethylaminopyridine, N'-methylimidazole and tetrazole. A hydroxyl group may be then substituted again for the dimethoxytrityl group in the dimethoxytrityl resin to provide for the addition of other nucleotides to the resin support. The procedure may be repeated to provide polynucleotides with extended chains of nucleotides.
Inventors
- 👤 Keiichi Itakura
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 13:59
CPC Classifications
C; C07; C07H; C07H21/00; C08; C08F; C08F8/00; C08F8/40
Claims Analysis
Total Claims: 59
Claim 1
Independent Claim
50 words
A method of forming a polynucleotide, including the following steps: providing a resin support, phosphorylating a nucleoside to provide a monotriazolide, combining the monotriazolide with the resin support to provide a dimethoxytrityl resin, and substituting a hydroxyl group for the dimethoxytrityl group in the dimethoxytrityl resin to provide the polynucleotide.
Claim 2
Independent Claim
50 words
A method of forming a polynucleotide, including the following steps: providing a resin support, phosphorylating a nucleoside to provide a monotriazolide, combining the monotriazolide with the resin support to provide a dimethoxytrityl resin, and substituting a hydroxyl group for the dimethoxytrityl group in the dimethoxytrityl resin to provide the polynucleotide.
Claim 3
Dependent Claim
48 words
The method set forth in claim 1 wherein the nucleoside is a 3'-hydroxyl nucleoside and the 3'-hydroxyl nucleoside is phosphorylated with a phosphoroditriazolide to obtain the monotriazolide and the monotriazolide is combined with the resin support in the presence of a nucleophilic catalyst to provide the dimethoxytrityl resin.
Claim 4
Dependent Claim
32 words
The method set forth in claim 3 wherein the nucleophilic catalyst is selected from a group consisting of dimethylaminopyridine, N'-methylimidazole and tetrazole and the resin in the resin support is a polystyrene.
Claim 5
Dependent Claim
23 words
The method set forth in claim 3 wherein the hydroxyl group is substituted for the dimethoxytrityl group in a solution of benzenesulfonic acid.
Claim 6
Dependent Claim
30 words
The method set forth in claim 1 wherein the resin support is obtained by forming an amino benzyl resin and combining this resin with an activated ester of a nucleoside.
Claim 7
Independent Claim
60 words
A method of forming a polynucleotide, including the following steps: providing a resin support, phosphorylating a 3'-hydroxyl nucleoside in a phosphoroditriazolide to form a monotriazolide, combining the monotriazolide and the resin support in the presence of a nucleophilic catalyst to provide a dimethoxytrityl resin, and removing the dimethoxytrityl group from the dimethoxytrityl resin in the presence of a suitable agent.
Claim 8
Independent Claim
60 words
A method of forming a polynucleotide, including the following steps: providing a resin support, phosphorylating a 3'-hydroxyl nucleoside in a phosphoroditriazolide to form a monotriazolide, combining the monotriazolide and the resin support in the presence of a nucleophilic catalyst to provide a dimethoxytrityl resin, and removing the dimethoxytrityl group from the dimethoxytrityl resin in the presence of a suitable agent.
Claim 9
Dependent Claim
16 words
The method set forth in claim 6 wherein the dimethoxytrityl resin is treated with benzenesulphonic acid.
Claim 10
Dependent Claim
43 words
The method set forth in claim 5 wherein the phosphorylation of the 3'-hydroxyl nucleoside is performed in dioxane or tetrafurane and the nucleophilic agent in the step of providing the dimethoxytrityl resin is selected from a group consisting of dimethylaminopyridine, N'-methylimidazole and tetrazole.
Claim 11
Dependent Claim
21 words
The combination set forth in claim 8 wherein the dimethylaminopyridine is mixed with tetrahydrofurane in the production of the dimethoxytrityl resin.
Claim 12
Dependent Claim
31 words
The method set forth in claim 9 wherein the resin support system is obtained by forming an amino hydroxyl resin and combining this resin with an activated ester of a nucleoside.
Claim 13
Dependent Claim
79 words
A method set forth in claim 9 wherein the resin support is obtained by treating chloromethyl polystyrene with potassium pthalimide to obtain a pthalimidomethyl-resin and the pthalimidomethyl-resin is converted into the amino-resin by treatment with hydrazine in ethanol and a 5'-O-dimethoxytrityl deoxynucleotide is reacted with succinic anhydride to form a monosuccinate derivative and the monosuccinate derivative is treated with pentachlorophenol to form a pentachlorophenol-treated nucleoside, the amino-resin is combined with the pentachlorophenol-treated nucleoside to obtain an amino-bonded resin support.
Claim 14
Dependent Claim
59 words
In a method of producing a polynucleotide, the steps of: providing a resin support phosphorylating a 3'-hydroxyl nucleoside in phosphoroditriazolide in the presence of a material selected from a group consisting of dioxane and hydrofurane to form a monotriazolide, and combining the monotriazolide and the resin support in the presence of a nucleophilic catalyst to provide a dimethoxytrityl resin.
Claim 15
Dependent Claim
59 words
In a method of producing a polynucleotide, the steps of: providing a resin support phosphorylating a 3'-hydroxyl nucleoside in phosphoroditriazolide in the presence of a material selected from a group consisting of dioxane and hydrofurane to form a monotriazolide, and combining the monotriazolide and the resin support in the presence of a nucleophilic catalyst to provide a dimethoxytrityl resin.
Claim 16
Dependent Claim
24 words
In the method set forth in claim 12, the nucleophilic catalyst constituting a material selected from a group consisting of dimethylaminopyridine, N'-methylimidazole and tetrazole.
Claim 17
Dependent Claim
13 words
In the method set forth in claim 13, the phosphoroditriazolide constituting O-chlorophenyl phosphoroditriazolide.
Claim 18
Dependent Claim
16 words
In the method set forth in claim 12, the nucleophilic catalyst consisting of dimethylaminopyridine in tetrafurane.
Claim 19
Dependent Claim
16 words
In the method set forth in claim 12, the resin in the resin support constituting polystyrene.
Claim 20
Dependent Claim
50 words
In a method of producing a polynucleotide, the steps of: providing a resin-supported nucleoside, combining a monotriazolide and the resin-supported nucleoside in the presence of a nucleophilic catalyst to provide a dimethoxytrityl resin, and substituting the dimethoxytrityl group in the dimethoxytrityl resin with a hydroxyl group to form the polynucleotide.
Claim 21
Dependent Claim
50 words
In a method of producing a polynucleotide, the steps of: providing a resin-supported nucleoside, combining a monotriazolide and the resin-supported nucleoside in the presence of a nucleophilic catalyst to provide a dimethoxytrityl resin, and substituting the dimethoxytrityl group in the dimethoxytrityl resin with a hydroxyl group to form the polynucleotide.
Claim 22
Dependent Claim
22 words
In the method set forth in claim 17, the nucleophilic catalyst being selected from a group consisting of dimethylaminopyridine, N'-methylimidazole and tetrazole.
Claim 23
Dependent Claim
15 words
In the method set forth in claim 17, the nucleophilic catalyst constituting dimethylaminopyridine in tetrafurane.
Claim 24
Dependent Claim
31 words
In the method set forth in claim 17, the substitution of the hydroxyl group for the dimethoxytrityl group in the dimethoxytrityl resin occurring in a solution of benzenesulfonic acid in chloroformmethanol.
Claim 25
Dependent Claim
51 words
In the method set forth in claim 20, the nucleophilic agent being selected from a group consisting of dioxane, dimethylaminopyridine or a combination of dimethylaminopyridine and tetrahydrofurane and the substitution of the hydroxyl group for the 3'-dimethoxytrityl group in the dimethoxytrityl resin occurring in a solution of benzenesulfonic acid in chloroformamethanol.
Claim 26
Dependent Claim
22 words
In the method set forth in claim 17, the nucleophilic catalyst being selected from a group consisting of dimethylaminopyridine, N'-methylimidazole and tetrazole.
Claim 27
Dependent Claim
21 words
In a method of producing a polynucleotide, the steps of: phosphorylating a 3'-hydroxyl nucleoside with a phosphoroditriazolide to provide a monotriazolide.
Claim 28
Dependent Claim
21 words
In a method of producing a polynucleotide, the steps of: phosphorylating a 3'-hydroxyl nucleoside with a phosphoroditriazolide to provide a monotriazolide.
Claim 29
Dependent Claim
30 words
The method set forth in claim 23 wherein the phosphoditriazolide is a O-chlorophenyl phosphoditriazolide and the phosphorylation occurs in a material selected from a group consisting of dioxane and tetrahydorfurane.
Claim 30
Independent Claim
73 words
A method of forming a polynucleotide, including the following steps: (a) providing an amino-resin, (b) providing a chlorophenolated nucleoside, (c) combining the amino-resin with the chlorophenolated nucleoside to obtain an amino-bonded resin support, (d) phosphorylating a nucleoside to obtain a monotriazolide, (e) combining the monotriazolide with the amino-bonded resin support to provide a dimethoxytrityl resin, and (f) substituting a hydroxyl group for the dimethoxytrityl group in the dimethoxytrityl resin to provide a polynucleotide.
Claim 31
Independent Claim
73 words
A method of forming a polynucleotide, including the following steps: (a) providing an amino-resin, (b) providing a chlorophenolated nucleoside, (c) combining the amino-resin with the chlorophenolated nucleoside to obtain an amino-bonded resin support, (d) phosphorylating a nucleoside to obtain a monotriazolide, (e) combining the monotriazolide with the amino-bonded resin support to provide a dimethoxytrityl resin, and (f) substituting a hydroxyl group for the dimethoxytrityl group in the dimethoxytrityl resin to provide a polynucleotide.
Claim 32
Dependent Claim
21 words
The method set forth in claim 25 wherein steps (c) and (d) are repeated to add additional nucleotides to the polynucleotide.
Claim 33
Dependent Claim
34 words
The method set forth in claim 25 wherein the phosphorylation in step (c) is provided by combining the nucleoside with a phosphoroditriazolide and step (d) is performed in the presence of a nucleophilic agent.
Claim 34
Dependent Claim
37 words
The method set forth in claim 27 wherein the phosphorylation in step (c) is performed in dioxane or tetrafurane and the nucleophilic agent in step (d) is selected from a group consisting of dimethylaminopyridine, N'-methylimidazole and tetrazole.
Claim 35
Dependent Claim
13 words
The method set forth in claim 26 wherein the resin is a polystyrene.
Claim 36
Dependent Claim
14 words
The method set forth in claim 26 wherein the resin is a silica gel.
Claim 37
Independent Claim
95 words
A method of forming a polynucleotide, including the following steps: (a) treating chloro-methyl polystyrene with potassium pthalimide to obtain a phthalimidomethyl-resin, (b) treating the phthalimidomethyl-resin with hydrazine in ethanol to obtain an amino-resin, (c) reacting the amino-resin with a pentachlorophenolated nucleoside to obtain an amide-bonded resin, (d) phosphorylating the nucleoside in a phosphoroditriazolide to form a monotriazolide, (e) combining the monotriazolide and the amide-bonded resin support in the presence of a nucleophilic agent to provide a dimethoxytrityl resin, and (f) removing the dimethoxytrityl group from the dimethoxytrityl resin in the presence of a suitable agent.
Claim 38
Independent Claim
95 words
A method of forming a polynucleotide, including the following steps: (a) treating chloro-methyl polystyrene with potassium pthalimide to obtain a phthalimidomethyl-resin, (b) treating the phthalimidomethyl-resin with hydrazine in ethanol to obtain an amino-resin, (c) reacting the amino-resin with a pentachlorophenolated nucleoside to obtain an amide-bonded resin, (d) phosphorylating the nucleoside in a phosphoroditriazolide to form a monotriazolide, (e) combining the monotriazolide and the amide-bonded resin support in the presence of a nucleophilic agent to provide a dimethoxytrityl resin, and (f) removing the dimethoxytrityl group from the dimethoxytrityl resin in the presence of a suitable agent.
Claim 39
Dependent Claim
23 words
The method set forth in claim 31 wherein steps (d), (e) and (f) are repeated to attach additional nucleosides to the amide-bonded resin.
Claim 40
Dependent Claim
16 words
The combination set forth in claim 32 wherein step (d) is performed in dioxane or tetrafurane.
Claim 41
Dependent Claim
22 words
The method set forth in claim 32 wherein the nucleophilic agent is selected from a group consisting of dimethylaminopyridine, N'-methylimidazole and tetrazole.
Claim 42
Dependent Claim
16 words
The method set forth in claim 33 wherein the nucleophilic agent is dimethylaminopyridine mixed with tetrahydrofurane.
Claim 43
Dependent Claim
11 words
The polynucleotide produced by the method set forth in claim 1.
Claim 44
Dependent Claim
11 words
The polynucleotide produced by the method set forth in claim 3.
Claim 45
Dependent Claim
11 words
The polynucleotide produced by the method set forth in claim 6.
Claim 46
Dependent Claim
12 words
The dimethoxytrityl resin produced by the method set forth in claim 12.
Claim 47
Dependent Claim
11 words
The polynucleotide produced by the method set forth in claim 25.
Claim 48
Dependent Claim
7 words
The polynucleotide set forth in claim 29.
Claim 49
Dependent Claim
11 words
The polynucleotide produced by the method set forth in claim 31.
Claim 50
Dependent Claim
13 words
The method set forth in claim 3 wherein the resin is silica gel.
Claim 51
Dependent Claim
13 words
The resin support for a polynucleotide comprising an amide bonded polystyrene resin support.
Claim 52
Dependent Claim
13 words
The resin support for a polynucleotide comprising an amide bonded polystyrene resin support.
Claim 53
Dependent Claim
13 words
In the method set forth in claim 17, the nucleophilic catalyst constituting dioxane.
Claim 54
Dependent Claim
15 words
In the method set forth in claim 19, the resin constituting polystyrene or silica gel.
Claim 55
Dependent Claim
15 words
In the method set forth in claim 44, the resin constituting polystyrene or silica gel.
Claim 56
Dependent Claim
20 words
The method set forth in claim 23 wherein the monotriazolide is coupled to a nucleoside to provide a dimethoxytrityl deoxynucleotide.
Claim 57
Dependent Claim
53 words
The method set forth in claim 17 wherein the resin support is provided by the following steps: (a) treating chloro-methyl polystyrene with potassium pthalimide to obtain a phthalimidomethyl-resin, (b) treating the phthalimidomethyl-resin with hydrazine in ethanol to obtain an amino-resin, (c) reacting the amino-resin with a pentachlorophenolated nucleoside to obtain an amino-bonded resin.
Claim 58
Dependent Claim
9 words
The polynucleotide produced by the method of claim 17.
Claim 59
Dependent Claim
9 words
The polynucleotide produced by the method of claim 46.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1986-08-29 | 1987-02-28 | 1987-08-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-08-29 | 1991-02-28 | 1991-08-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-08-29 | 1995-02-28 | 1995-08-30 | $7700.00 | Window Closed / Lapsed |