Solid-phase synthesis of polynucleotides

• US 4,373,071 A
• Filed: 04/30/1981
• Issued: 02/08/1983
• Est. Priority Date: 04/30/1981
• Status: Expired due to Term

• Current Assignee: Edwards S Irons PC, Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: City of Hope Research Institute
• Inventors: Itakura, Keiichi
• Primary Examiner(s): Michl, Paul R.
• Application Number:
  US06/258,925
• Time in Patent Office: 649 Days
• Field of Search: 525/366, 525/340, 525/375, 521/32, 536/22, 536/23, 536/24, 536/25, 536/26, 536/27, 536/28, 536/29
• US Class Current: 525/375
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C08F 112/08 Styrene
• CPC Class Codes: C08F 8/18 Introducing halogen atoms or halogen-containing groups
• CPC Class Codes: C08F 8/30 Introducing nitrogen atoms or nitrogen-containing groups
• CPC Class Codes: C08F 8/32 by reaction with amines

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.

Claims

1. 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.

2. 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.

3. The method set forth in claim 2 wherein

the resin is selected from a group consisting of silica gel and polystyrene.

4. 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.

5. 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.

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

14. 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.

15. 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.

16. 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.

31. 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.

32. A method as set forth in claim 31 wherein

steps (d) and (e) are repeated to couple additional nucleotides in the polynucleotide.

33. 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.

34. 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.

35. 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.

36. 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.

37. 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.

17. 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.

21. 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.

22. A method as set forth in claim 17 wherein the resin support is a polyacrylmorpholide.

18. 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.

19. 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.

20. 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.

23. 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.

24. 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.

25. 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.

26. 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.

27. 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.

28. 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.

29. 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.

30. 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.

38. 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.

39. A method as set forth in claim 38 wherein

the dimethoxytrityl group in the resin support is hydroxylated by treatment with benzenesulphonic acid.

40. 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.

41. 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.

42. 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.

43. 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.

44. 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.