Process for the synthesis of oligonucleotides
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IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C07H-021/02
C07H-021/04
출원번호
US-0853662
(2007-09-11)
등록번호
US-8138330
(2012-03-20)
발명자
/ 주소
Leuck, Michael
Wolter, Andreas
Stumpe, Alfred
출원인 / 주소
Sigma-Aldrich Co. LLC
대리인 / 주소
Polsinelli Shughart PC
인용정보
피인용 횟수 :
0인용 특허 :
12
초록▼
The present invention discloses novel methods for the synthesis of oligonucleotides with nucleoside phosphoramidites on solid supports. The methods comprise the stepwise chain assembly of oligonucleotides on supports with 5′-acyl phosphoramidites. The synthesis cycles consist of a front end deprotec
The present invention discloses novel methods for the synthesis of oligonucleotides with nucleoside phosphoramidites on solid supports. The methods comprise the stepwise chain assembly of oligonucleotides on supports with 5′-acyl phosphoramidites. The synthesis cycles consist of a front end deprotection step which is conducted with a solution of a primary amine or a phenolate, a phosphoramidite coupling step with a 5′-acyl nucleoside phosphoramidite in the presence of an activator, a phosphite oxidation step and an optional capping step. The novel methods improve the quality of synthetic oligonucleotides due to the irreversibility of the front end deprotection step, which prevents the formation of deletion sequences, and due to the avoidance of acidic reagents in the synthesis cycles, which prevent the formation of depurination side products. The invention further discloses novel nucleoside phosphoramidite compositions wherein the phosphoramidites carry acyl front end protective groups which are cleavable with primary amines or phenolates. The invention is applicable to the synthesis of oligodeoxyribonucleotides, oligoribonucleotides and oligonucleotides with modifications in their sugar or phosphate groups.
대표청구항▼
1. A method for the solid phase synthesis of oligonucleotides, wherein each synthesis cycle of the method comprises: a) cleaving an acyl front end protective group from a nucleoside immobilized on a solid support to liberate a hydroxyl group on the nucleoside, the acyl front end protective group com
1. A method for the solid phase synthesis of oligonucleotides, wherein each synthesis cycle of the method comprises: a) cleaving an acyl front end protective group from a nucleoside immobilized on a solid support to liberate a hydroxyl group on the nucleoside, the acyl front end protective group comprising either an electron withdrawing group or a group capable of neighboring group participation in close proximity to its carbonyl group such that the acyl group is cleavable in ten minutes or less at room temperature by a primary amine, a secondary amine, or a mixture thereof; andb) coupling an acyl protected nucleoside phosphoramidite with the hydroxyl group of the nucleoside immobilized on the solid support. 2. The method of claim 1, wherein the acyl protective group is selected from the group consisting of an oxalyl monoester group, an ortho-phenoxycarbonyl benzoyl group, and a cyclohexyloxydicarbonyl group. 3. The method of claim 2, wherein the ortho-phenoxycarbonyl benzoyl group is selected from the group consisting of a 2-(4-nitrophenoxy)carbonyl benzoyl group and a 2-(2-cyanophenoxy)carbonyl benzoyl group. 4. The method of claim 1, wherein the acyl protective group is attached to the 5′-hydroxyl function of the nucleoside. 5. The method of claim 1, wherein the acyl protective group is attached to the 3′-hydroxyl function of the nucleoside. 6. The method of claim 1, wherein the cleaving reagent comprises a primary amine. 7. The method of claim 6, wherein the primary amine is selected from the group consisting of n-butylamine, n-hexylamine, 2-methoxyethylamine, and 2-(N,N-diethylamino)ethylamine. 8. The method of claim 1, wherein the cleavage of the front end protective group occurs in 1 minute or less at room temperature. 9. The method of claim 1, wherein the first nucleoside of the oligonucleotide to be synthesized is attached to the solid support via an acid-labile linker. 10. The method of claim 1, wherein the base moiety of the nucleoside phosphoramidite is either unprotected or protected with an acid-labile nucleobase protective group. 11. The method of claim 10, wherein, after synthesis of the oligonucleotide, the nucleobase protective groups of the oligonucleotide are removed with an acidic reagent. 12. The method of claim 11, wherein the acidic reagent is aqueous. 13. The method of claim 12, wherein the aqueous acidic reagent has a pH in the range of pH 2 to pH 4. 14. The method of claim 1, wherein the nucleoside phosphoramidite is a ribonucleoside and the 2′-hydroxyl function of the ribonucleoside is protected with an acid-labile 2′-protective group. 15. A nucleoside phosphoramidite comprising an acyl front end protective group and a phosphoramidite group, wherein the acyl front end protective group comprises either an electron withdrawing group or a group capable of neighboring group participation in close proximity to its carbonyl group such that the acyl group is cleavable with a primary amine, a secondary amine, or a mixture thereof in 10 minutes or less at room temperature. 16. The nucleoside phosphoramidite of claim 15, wherein the acyl front end protective group is an oxalyl monoester group. 17. The nucleoside phosphoramidite of claim 15, wherein the acyl front end protective group is an ortho-phenoxycarbonyl benzoyl group. 18. The nucleoside phosphoramidite of claim 17, wherein the ortho-phenoxycarbonyl benzoyl group is selected from the group consisting of a 2-(4-nitrophenoxy)carbonyl benzoyl group and a 2-(2-cyanophenoxy)carbonyl benzoyl group. 19. The nucleoside phosphoramidite of claim 15, wherein the acyl front end protective group is a cyclohexyloxydicarbonyl group. 20. The nucleoside phosphoramidite of claim 15, wherein the phosphoramidite group comprises a β-cyanoethyl phosphate protective group. 21. The nucleoside phosphoramidite of claim 15, wherein the base moiety of the nucleoside phosphoramidite is protected with an acid-labile nucleobase protective group. 22. The nucleoside phosphoramidite of claim 15, wherein the nucleoside is a ribonucleoside and the 2′-hydroxyl function of the ribonucleoside is protected with an acid-labile 2′-protective group. 23. The nucleoside phosphoramidite of claim 22, wherein the acid-labile 2′-protective group a 4-methoxytetrahydro-pyranyl group.
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이 특허에 인용된 특허 (12)
Dover, Lance, Configuring integrated circuit devices in a data processing system.
Dellinger Douglas J. ; Caruthers Marvin H. ; Betley Jason R.,GBX, Solid phase synthesis of oligonucleotides using carbonate protecting groups and alpha-effect nucleophile deprotection.
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