US4373071A
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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: 1983-02-08
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Expires: 2001-04-30
Abstract
Abstract A commercially available resin such as polystyrene is converted into a phthalimidomethyl-resin by treatment with potassium phthalimide. The phthalimidomethyl-resin is converted into an amino resin with hydrazine in ethanol. The amino resin is then combined with an activated ester of a nucleoside to obtain an amide-bonded dimethoxytrityl resin. The activated ester of the nucleoside is obtained by reacting a nucleoside with succinic anhydride in the presence of 4-(dimethylamino) pyridine in pyridine to provide a monosuccinate derivative, which is in turn treated with pentachlorophenol and dicyclohexylcarbodimide in dimethylformamide. Any unreacted amino groups in the amide-bonded dimethoxytrityl resin may be masked and the dimethoxytrityl group may be removed by treatment of the amide-bonded resin with a solution of benzenesulfonic acid to obtain a hydroxylated resin support. Additional mononucleotides, dinucleotides and trinucleotides may be added to the resin support in the presence of a coupling reagent such as 2,4,6-triisopropylbenzenesulfonyl tetrazolide. Any unreacted 5'-hydroxyl group may be masked with acetic anhydride. The steps described in the previous two sentences may be repeated to form polynucleotides of any desired sequence. The resin may also be a polyacrylmorpholide. This resin may be converted into an amino resin with ethylene diamine in ethylene glycol. The resin may also be a silica gel. The silica gel may be treated by any of the above methods, to provide a solid-phase resin support for the nucleotides.
Inventors
- 👤 Keiichi Itakura
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 16:21
CPC Classifications
C; C07; C07H; C07H21/00; C08; C08F; C08F8/00; C08F8/18; C08F8/30; C08F8/32
Claims Analysis
Total Claims: 50
Claim 1
Independent Claim
45 words
In a method of creating polynucleotides, the following steps: treating a resin with potassium phthalimide to obtain a phthalimidomethyl-resin, converting the phthalimidomethyl-resin into an amino resin with hydrazine, and combining the amino-resin with an activated ester of a nucleoside to obtain an amino-bonded dimethoxytrityl resin.
Claim 2
Dependent Claim
45 words
In a method of creating polynucleotides, the following steps: treating a resin with potassium phthalimide to obtain a phthalimidomethyl-resin, converting the phthalimidomethyl-resin into an amino resin with hydrazine, and combining the amino-resin with an activated ester of a nucleoside to obtain an amino-bonded dimethoxytrityl resin.
Claim 3
Dependent Claim
36 words
The method set forth in claim 1 wherein the activated ester of the nucleoside is obtained by reacting a nucleoside with succinic anhydride to provide a monosuccinic derivative and by treating the monosuccinic derivative with pentachlorophenol.
Claim 4
Dependent Claim
21 words
The method set forth in claim 2 wherein the resin is selected from a group consisting of silica gel and polystyrene.
Claim 5
Dependent Claim
48 words
The method set forth in claim 3 wherein the nucleoside is reacted with succinic anhydride in the presence of 4-dimethylamino pyridine to provide the monosuccinic derivative and the monosuccinic derivative is treated with pentachloraphenol and dicyclohexyl carbodimide in dimethyl formamide to obtain the activated ester of the nucleoside.
Claim 6
Independent Claim
50 words
A method of creating polynucleotides, including the following steps; converting a resin into an amino-resin; combining the amino-resin with a chlorophenolated nucleoside to obtain an amino-bonded dimethoxytrityl resin support, and hydroxylating the dimethoxytrityl group in the amino-bonded resin support to provide for the coupling of nucleotides to the resin support.
Claim 7
Independent Claim
50 words
A method of creating polynucleotides, including the following steps; converting a resin into an amino-resin; combining the amino-resin with a chlorophenolated nucleoside to obtain an amino-bonded dimethoxytrityl resin support, and hydroxylating the dimethoxytrityl group in the amino-bonded resin support to provide for the coupling of nucleotides to the resin support.
Claim 8
Dependent Claim
49 words
The method set forth in claim 5 wherein additional nucleotides are added to the hydroxylated amino-bonded resin support in the presence of a coupling reagent and any unreacted 5'-hydroxyl group in the amino-bonded resin support is masked before the additional nucleotides are added to the hydroxylated amino-bonded resin support.
Claim 9
Dependent Claim
52 words
The method set forth in claim 6 wherein the coupling reagent for adding the nucleotides to the hydroxylated amino-bonded resin support is 2,4,6-triisopropylbenzenesulfonyl tetrazolide and and any unreacted 5'-hydroxyl group in the amino-bonded resin support is masked with acetic anhydride before the additional nucleotides are added to the hydroxylated amino-bonded resin support.
Claim 10
Dependent Claim
43 words
The method set forth in claim 7 wherein the resin is selected from a group consisting of silica gel and polystyrene and the resin is treated with potassium phthalimide to obtain a phthalimidomethyl-resin and the phthalimidomethyl-resin is converted into the amino-resin with hydrazine.
Claim 11
Dependent Claim
24 words
The method set forth in claim 7 wherein the dimethoxytrityl group in the amino-bonded resin support is hydroxylated with a solution of benezenesulphonic acid.
Claim 12
Dependent Claim
26 words
The method set forth in claim 6 wherein the resin constitutes a polyacrylmorpholide and the resin is converted into the amino-resin with ethylenediamine in ethylene glycol.
Claim 13
Independent Claim
55 words
A method of creating a polynucleotide, including the following steps: (a) providing a resin, (b) applying chemicals containing amino radicals to the resin to obtain an amino-resin, (c) converting the amino-resin into an amino-bonded dimethoxytrityl resin support, (d) hydroxylating the dimethoxytrityl group in the resin support, and (e) coupling nucleotides to the hydroxylated resin support.
Claim 14
Independent Claim
55 words
A method of creating a polynucleotide, including the following steps: (a) providing a resin, (b) applying chemicals containing amino radicals to the resin to obtain an amino-resin, (c) converting the amino-resin into an amino-bonded dimethoxytrityl resin support, (d) hydroxylating the dimethoxytrityl group in the resin support, and (e) coupling nucleotides to the hydroxylated resin support.
Claim 15
Dependent Claim
36 words
A method as set forth in claim 11 wherein the nucleotides are added to the hydroxylated resin support in the presence of a coupling reagent and any unreacted 5'-hydroxyl group in the resin support is masked.
Claim 16
Dependent Claim
25 words
A method set forth in claim 12 wherein the steps (d) and (e) of claim 12 are repeated to couple additional nucleotides to the polynucleotide.
Claim 17
Dependent Claim
39 words
A method set forth in claim 13 wherein the coupling reagent is 2,4,6-triisopropyl benzenesulfonyl tetrazolide and any unreacted 5'-hydroxyl group in the amino-bonded resin support is masked with acetic anhydride before the nucleotides are coupled to the resin support.
Claim 18
Dependent Claim
29 words
A method set forth in claim 13 wherein the resin is a polyacrylmorpholide and the resin is converted to the amino-resin with ethylenediamine in the presence of ethylene glycol.
Claim 19
Dependent Claim
47 words
A method as set forth in claim 13 wherein the resin is selected from a group consisting of silica gel and polystyrene, and the resin is converted into a phthalimidomethyl-resin by treatment with potassium phthalimide, and the phthalimidomethyl-resin is converted to the amino-resin with hydrazine in ethanol.
Claim 20
Dependent Claim
50 words
A method as set forth in claim 17 wherein the amino-bonded resin support is treated with a solution of benzenesulphonic acid to hydroxylate the dimethoxytrityl group after the masking of the unreacted 5'-hydroxyl group in the amino-bonded resin support and before the nucleotides are coupled to the hydroxylated resin support.
Claim 21
Independent Claim
37 words
A method of creating polynucleotides, including the following steps: providing a resin support, providing an activated ester of a nucleoside, and combining the activated ester of the nucleoside and the resin support to obtain a resin-supported nucleoside.
Claim 22
Independent Claim
37 words
A method of creating polynucleotides, including the following steps: providing a resin support, providing an activated ester of a nucleoside, and combining the activated ester of the nucleoside and the resin support to obtain a resin-supported nucleoside.
Claim 23
Dependent Claim
33 words
A method as set forth in claim 18, including the following steps: hydroxylating the dimethoxytrityl group in the resin-supported nucleoside and adding nucleotides to the hydroxylated resin-supported nucleoside to obtain a resin-supported polynucleotide.
Claim 24
Dependent Claim
45 words
A method as set forth in claim 19 wherein the dimethoxytrityl group in the resin-supported nucleotide is hydroxylated in the presence of a solution of a benzenesulfonic acid and wherein the nucleotides are added to the hydroxylated resin support in the presence of 2,4,6-triisopropylbenzenesulfonyl tetrazolide.
Claim 25
Dependent Claim
26 words
A method as set forth in claim 17 wherein the resin in the resin support is selected from a group consisting of silica gel and polystyrene.
Claim 26
Dependent Claim
15 words
A method as set forth in claim 17 wherein the resin support is a polyacrylmorpholide.
Claim 27
Dependent Claim
44 words
A method set forth in claim 20 wherein the activated ester of a nucleoside is provided by reacting a nucleoside with a succinic anhydride to provide a monosuccinate derivative and treating the monosuccinate derivative with pentachlorophenol to produce the activated ester of the nucleoside.
Claim 28
Independent Claim
52 words
A method of creating polynucleotides, including the following steps: providing a resin, converting the resin into an amino-resin, providing a nucleoside, combining the amino-resin and the nucleoside to obtain an amino-bonded dimethoxytrityl resin support, hydroxylating the dimethoxytrityl group in the amino-bonded resin support, and attaching nucleotides to the hydroxylated amino-bonded resin support.
Claim 29
Independent Claim
52 words
A method of creating polynucleotides, including the following steps: providing a resin, converting the resin into an amino-resin, providing a nucleoside, combining the amino-resin and the nucleoside to obtain an amino-bonded dimethoxytrityl resin support, hydroxylating the dimethoxytrityl group in the amino-bonded resin support, and attaching nucleotides to the hydroxylated amino-bonded resin support.
Claim 30
Dependent Claim
25 words
A method set forth in claim 24 wherein the resin is selected from a group consisting of a polyacrylmorpholide, a polystyrene and a silica gel.
Claim 31
Dependent Claim
47 words
A method set forth in claim 25 wherein the dimethoxytrityl group in the amide-bonded resin support is hydroxylated by treatment with a solution of a benzenesulphonic acid and the nucleotides are attached to the hydroxylated resin support in the presence of a coupling reagent constituting 2,4,6-triisopropylbenzenesulfonyl tetrazolide.
Claim 32
Dependent Claim
27 words
A method as set forth in claim 26 wherein the resin is a polyacrylmorpholide and the resin is converted into an amino-resin with ethylenediamine in ethylene glycol.
Claim 33
Dependent Claim
49 words
A method as set forth in claim 26 wherein the resin is selected from a group consisting of silica gel and a polystyrene and the resin is converted into a pthalimidomethyl-resin by treatment with potassium phthalimide and the phthalimidomethyl-resin is converted into the amino resin with hydrazine in ethanol.
Claim 34
Dependent Claim
41 words
A method as set forth in claim 26 wherein the resin is a silica gel and the resin is converted into a phthalimidomethyl-resin by treatment with potassium phthalimide and the phthalimidomethyl-resin is converted into the amino resin with hydrazine in ethanol.
Claim 35
Dependent Claim
33 words
A method as set forth in claim 24 wherein the nucleoside is converted into a chlorophenolated nucleoside and the amino-resin and the chlorophenolated resin are combined to obtain the amide-bonded dimethoxytrityl resin support.
Claim 36
Dependent Claim
65 words
A method as set forth in claim 11 wherein steps (c) is provided as follows: (f) providing a deoxynucleoside, (g) treating the deoxynucleoside with succinic anhydride to provide a monosuccinate derivative, (h) treating the monosuccinate derivative with pentachlorophenol to form an activated ester of the nucleoside, and (i) combining the amino-resin and the activated ester of the nucleoside to obtain the amino-bonded dimethoxytrityl resin support.
Claim 37
Dependent Claim
22 words
A method as set forth in claim 31 wherein steps (d) and (e) are repeated to couple additional nucleotides in the polynucleotide.
Claim 38
Dependent Claim
31 words
A method as set forth in claim 31 wherein the resin is a polyacrylmorpholide and the resin is converted to the amino-resin with ethylene diamine in the presence of ethylene glycol.
Claim 39
Dependent Claim
47 words
A method as set forth in claim 31 wherein the resin is selected from a group consisting of silica gel and polystyrene, and the resin is converted into a pthalimidomethyl-resin by treatment with potassium phthalimide and the phthalimidomethyl-resin is converted to the amino-resin with hydrazine in ethanol.
Claim 40
Dependent Claim
27 words
A method as set forth in claim 33 wherein step (e) is provided by adding nucleotides to the hydroxylated resin support in the presence of 2,4,6-triisopropylbenezenesulfonyl tetrazolide.
Claim 41
Dependent Claim
27 words
A method as set forth in claim 34 wherein step (e) is provided by adding nucleotides to the hydroxylated resin support in the presence of 2,4,6-triisopropylbenezenesulfonyl tetrazolide.
Claim 42
Dependent Claim
48 words
A method as set forth in claim 31 wherein any unreacted 5'-hydroxyl group in the amino-bonded resin support is masked with acetic anhydride before the nucleotides are coupled to the resin support and the nucleotides are added to the hydroxylated resin support in the presence of 2,4,6-triisopropylbenezenesulfonyl tetrazolide.
Claim 43
Independent Claim
80 words
A method of creating a polynucleotide, including the following: (a) providing a deoxynucleoside, (b) treating the deoxynucleoside with succinic anhydride to provide a monosuccinate derivative, (c) treating the monosuccinate derivative with pentachlorophenol to form an activated ester of the nucleoside, (d) providing an amino-resin (e) combining the amino-resin and the activated ester of the nucleoside to obtain an amino-bonded dimethoxytrityl resin support, (f) hydrozylating the dimethoxytrityl group in the resin support, and (g) coupling nucleotides to the hydroxylated resin support.
Claim 44
Independent Claim
80 words
A method of creating a polynucleotide, including the following: (a) providing a deoxynucleoside, (b) treating the deoxynucleoside with succinic anhydride to provide a monosuccinate derivative, (c) treating the monosuccinate derivative with pentachlorophenol to form an activated ester of the nucleoside, (d) providing an amino-resin (e) combining the amino-resin and the activated ester of the nucleoside to obtain an amino-bonded dimethoxytrityl resin support, (f) hydrozylating the dimethoxytrityl group in the resin support, and (g) coupling nucleotides to the hydroxylated resin support.
Claim 45
Dependent Claim
23 words
A method as set forth in claim 38 wherein the dimethoxytrityl group in the resin support is hydroxylated by treatment with benzenesulphonic acid.
Claim 46
Dependent Claim
28 words
A method as set forth in claim 38 wherein the nucleotides are coupled to the hydroxylated resin support in the presence of a coupling agent constituting 2,4,6-triisopropylbenezenesulfonyl tetrazolide.
Claim 47
Dependent Claim
29 words
A method as set forth in claim 38 wherein the resin in the amino-resin is selected from a group consisting of a polyacrylmorpholide, a polystyrene and a silica gel.
Claim 48
Dependent Claim
43 words
The method set forth in claim 40 wherein the dimethoxytrityl group in the resin support is hydroxylated by treatment with a benezenesulphonic acid and the nucleotides are coupled to the hydroxylated resin support in the presence of a coupling agent constituting 2,4,6-triisopropylbenezenesulfonyl tetrazolide.
Claim 49
Dependent Claim
48 words
The method set forth in claim 41 wherein the resin in the amino-resin is selected from a group consisting of polystyrene and silica gel and the resin is treated with potassium phthalimide to obtain a phthalimido methyl-resin and the phthalimido methyl-resin is converted into the amino-resin with hydrazine.
Claim 50
Dependent Claim
26 words
The method set forth in claim 42 wherein the resin constitutes a polyacrylmorphouide and the resin is converted into the amino-resin with ethylenediamine in ethylene glycol.
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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) | 1986-02-07 | 1986-08-09 | 1987-02-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-02-07 | 1990-08-09 | 1991-02-08 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-02-07 | 1994-08-09 | 1995-02-08 | $7700.00 | Window Closed / Lapsed |