Purification of triphosphorylated oligonucleotides using capture tags
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C07H-021/00
C07H-021/02
C07H-001/04
출원번호
US-0007752
(2012-03-28)
등록번호
US-9399658
(2016-07-26)
우선권정보
EP-11160032 (2011-03-28)
국제출원번호
PCT/EP2012/055520
(2012-03-28)
§371/§102 date
20130926
(20130926)
국제공개번호
WO2012/130886
(2012-10-04)
발명자
/ 주소
Ludwig, Janos
Goldeck, Marion
Sproat, Brian
출원인 / 주소
Rheinische Friedrich-Wilhelms-Universität Bonn
대리인 / 주소
Rothwell, Figg, Ernst & Manbeck, P.C.
인용정보
피인용 횟수 :
1인용 특허 :
34
초록
The present invention relates to a method of preparing triphosphate-modified oligonucleotides using a capture tag. The method allows the synthesis and purification of triphosphate-modified oligonucleotides in high yield and purity suitable for pharmaceutical applications.
대표청구항▼
1. A method of preparing an oligonucleotide of formula (I), wherein V1, V3 and V5 are independently in each case selected from O, S and Se;V2, V4 and V6 are independently in each case selected from OH, OR1, SH, SR1, F, NH2, NHR1, N(R1)2 and BH3−M+,W1 is O or S,W2 is O, S, NH or NR2,W3 is O, S, NH, N
1. A method of preparing an oligonucleotide of formula (I), wherein V1, V3 and V5 are independently in each case selected from O, S and Se;V2, V4 and V6 are independently in each case selected from OH, OR1, SH, SR1, F, NH2, NHR1, N(R1)2 and BH3−M+,W1 is O or S,W2 is O, S, NH or NR2,W3 is O, S, NH, NR2, CH2, CHHal or C(Hal)2,R1, R2 and R3 are selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 acyl or a cyclic group, each substituted or unsubstituted,or wherein two R1 may form a ring together with an N-atom bound thereto,M+ is a cation,X is NH, NR3, O or S,Z represents a capture tag which is a long-chain alkyl residue, a perfluoroalkyl entity, an azide or an alkynyl group,Y represents a bond or a linker connecting the capture tag to X, and ON represents an oligonucleotide comprising at least 4 nucleotide or nucleotide analogue building blocks,comprising the steps: (a) reacting a compound of formula (IIa) with an oxidizing agent to obtain a compound of formula (IIb) (b) reacting a compound of formula (IIb) with a capture tag agent of formula (III), Z—Y—XH (III)to obtain a reaction product comprising the oligonucleotide of formula (I), and(c) contacting the reaction product of step (b) with a capture reagent capable of interacting with the capture tag, wherein the contacting takes place under conditions which allow separation of the oligonucleotide (I) from other species contained in said reaction product. 2. The method of claim 1, wherein the capture tag and the capture reagent capable of interacting therewith are selected from: (i) a hydrophobic or fluorinated group and a chromatographic material with affinity for hydrophobic or fluorinated groups;(ii) a first partner of a non-covalent binding pair and a second partner of a non-covalent binding pair, and(iii) a first partner of a covalent binding pair and a second partner of a covalent binding pair, where the first and second partner form covalent bonds. 3. The method of claim 1, wherein the triphosphate/triphosphate analogue group is attached to the 5′-terminus of the oligonucleotide. 4. The method of claim 1, further comprising the step: (d) removing the capture tag to obtain an oligonucleotide of formula (IV):wherein V1, V3, V5, V2, V4, V6, W1, W2, W3 and ON are as defined in claim 1. 5. The method of claim 1, wherein the oligonucleotide is selected from desoxyribonucleotides, ribonucleotides and oligonucleotide analogues. 6. The method of claim 1, wherein the oligonucleotide is single-stranded or double stranded. 7. The method of claim 6, wherein the oligonucleotide is double-stranded and the duplex is closed by a loop at the distal end thereof, wherein the loop comprises nucleotide and/or non-nucleotide building blocks. 8. The method of claim 6, wherein the oligonucleotide is double-stranded and the duplex is blunt-ended at the proximal end thereof. 9. The method of claim 1, wherein the oligonucleotide comprises a cell-specific targeting entity covalently attached thereto. 10. The method of claim 1, wherein the oligonucleotide of formula (I) or an oligonucleotide of formula (IV) wherein V1, V3, V5, V2, V4, V6, W1, W2, W3 and ON are as defined in claim 1, is an activator of RIG-1. 11. Oligonucleotide of Formula (I), obtainable by a method comprising (a) reacting a compound of formula (IIa) with an oxidizing agent to obtain a compound of formula (IIb) (b) reacting a compound of formula (IIb) with a capture tag agent of formula (III), Z—Y—XH (III)to obtain a reaction product comprising the oligonucleotide of formula (I), and(c) contacting the reaction product of step (b) with a capture reagent capable of interacting with the capture tag, wherein the contacting takes place under conditions which allow separation of the oligonucleotide (I) from other species contained in said reaction product,wherein V1, V3 and V5 are independently in each case selected from O, S and Se; V2, V4 and V6 are independently in each case selected from OH, OR1, SH, SR1, F,NH2, NHR1, N(R1)2 and BH3−M+,W1 is O or S,W2 is O, S, NH or NR2,W3 is O, S, NH, NR2, CH2, CHHal or C(Hal)2,R1, R2 and R3 are selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 acyl or a cyclic group, each substituted or unsubstituted,or wherein two R1 may form a ring together with an N-atom bound thereto,M+ is a cation,X is NH, NR3, O or S,Z represents a capture tag which is a long-chain alkyl residue, a perfluoroalkyl entity, an azide or an alkynyl group,Y represents a bond or a linker connecting the capture tag to X, andON represents an oligonucleotide comprising at least 4 nucleotide or nucleotide analogue building blocks. 12. A kit for preparing an oligonucleotide of formula (I) wherein V1, V3, V5, V2, V4, V6, W1, W2, W3, X, Y, Z and ON are defined as in claim 1, wherein the kit comprises:(a) a capture tag agent of formula (III) Z—Y—XH (III) wherein X, Z and Y are defined as in claim 1, and(b) a capture reagent capable of interacting with the capture tag. 13. A modified oligonucleotide of formula (I) whereinX is NH, O, R—O—[P(V1)V2—W1]n or R—O—P(V3)V4—W2—P—(V1)V2—W1,n is 1-12,Y is a bond,Z is C13-C24 alkyl, Q or QNHC2-C24 alkyl,Q is C1-C24 alkyl,R is C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl and lipids, orR is C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C2-C24 acyl or a cyclic group, and is substituted or unsubstituted,and V1, V2, V3, V4, V5, V6, W1, W2, W3 and ON are defined in claim 1. 14. The modified oligonucleotide of claim 13, whereinX is NH or O, andZ is selected from the group consisting of C13-C24 alkyl, Q and QNHC2-C24 alkyl. 15. The modified oligonucleotide of claim 13, whereinX is R—O—[P(V1)V2—W1]n,n is 1 or 2, andV1, V2 and W1 are O. 16. The method according to claim 2, wherein said chromatographic material with affinity for hydrophobic or fluorinated groups is a reversed phase material or a fluorous affinity support. 17. The method of claim 3, wherein the triphosphate/triphosphate analogue group is attached to the 5′-OH-group of the 5′-terminal sugar thereof. 18. The modified oligonucleotide according to claim 13, wherein n is 1 or 2, and V1, V2, V3, V4, V5, V6, W1, W2 and W3 are O.
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