Method of preparing 5′-amino-linker oligonucleotides derivatives and analogous 5′-labeled-linker oligonucleotides therefrom
View Patent ↗A method for preparing amino linker oligonucleotides is provided. More specifically, a method of preparing 5′-amino-linker oligonucleotides comprising the steps of: introducing an amino linker having a protecting group into the 5′ terminus of an oligonucleotide; and removing the protecting group from the amino linker oligonucleotide by contacting with acetic acid and 2,2,2-trifluoroethanol is provided. The amino protecting group is efficiently removed from the amino linker oligonucleotides, and thereby achieving a high yield of the amino linker oligonucleotides.
1. A method of preparing 5′-amino linker oligonucleotides, comprising the steps of:
introducing an amino linker having a protecting group at the 5′ terminus of an oligonucleotide; and
removing the protecting group from the amino linker oligonucleotide by contacting with a mixture of acetic acid and trifluoroethanol (F 3 CCH 2 OH)
wherein the linker is a carbon chain having 1 to 30 carbons or polyethylene glycol presented by (CH 2 CH 2 O)n (n=2 to 400), and
the protecting group is selected from the group consisting of monomethoxytrityl (MMT), 6-(4,4′-Dimethoxy-4″-methylsulfonyl-trityl) (DMS(O)MT), 2-(Biophenyl-4-yl)propan-2-yloxycarbonyl (Bpoc), and 2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl (Ddz) groups.
2. The method according to claim 1 , wherein the mixture of acetic acid and trifluoroethanol (F 3 CCH 2 OH) is prepared by dissolving acetic acid and trifluoroethanol in an organic solvent with the volume ratio of acetic acid:trifluoroethanol:organic solvent being 1:1˜5:5˜10.
3. The method according to claim 2 , wherein the organic solvent is one or more selected from the group consisting of dichloromethane, dichloroethane, dimethyl formamide (DMF), acetonitrile (AcCN), dimethyl sulfoxide (DMSO), alcohol having 1 to 4 carbons, and N-methylpyrrolidinone (NMP).
4. The method according to claim 1 , wherein the protecting group is monomethoxytrityl (MMT).
5. The method according to claim 1 , further comprising the step of preparing said oligonucleotide by solid phase reaction prior to the step of introducing an amino linker with a protecting group into the 5′ terminus of an oligonucleotide.
6. A method of preparing 5′-labeled linker oligonucleotides comprising the steps of:
introducing an amino linker having a protecting group at the 5′ terminus of an oligonucleotide;
removing the protecting group from the amino linker oligonucleotide by contacting with a mixture of acetic acid and trifluoroethanol (F 3 CCH 2 OH); and
binding a labeling agent to the amino group of the amino linker oligonucleotides
wherein the linker is a carbon chain having 1 to 30 carbons or polyethylene glycol presented by (CH 2 CH 2 O)n (n=2 to 400), and
the protecting group is selected from the group consisting of monomethoxytrityl (MMT), 6-(4,4′-Dimethoxy-4″-methylsulfonyl-trityl) (DMS (O)MT), 2-(Biophenyl-4-yl)propan-2-yloxycarbonyl (Bpoc), and 2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl(Ddz) groups.
7. The method according to claim 6 , wherein the mixture of acetic acid and trifluoroethanol (F 3 CCH 2 OH) is prepared by dissolving acetic acid and trifluoroethanol in an organic solvent with the volume ratio of acetic acid:trifluoroethanol (F3CCH2OH):organic solvent being 1:1˜5:5˜10.
8. The method according to claim 7 , wherein the organic solvent is one or more kinds selected from the group consisting of dichloromethane, dichloroethane, dimethyl formamide (DMF), acetonitrile (AcCN), dimethyl sulfoxide (DMSO), alcohol of carbon number 1 to 4, and N-methylpyrrolidinone (NMP).
9. The method according to claim 6 , wherein the protecting group is selected from the group consisting of monomethoxytrityl (MMT).
10. The method according to claim 6 , further comprising the step of preparing the oligonucleotide by solid phase reaction prior to the step of introducing an amino linker with a protecting group into the 5′ terminus of an oligonucleotide.
11. The method according to claim 6 , wherein the labeling agent is one or more selected from the group consisting of a carboxylic acid, a chloroformate, a thiol anhydride, an isocyanate, and a chelate having a functional group capable of binding to an amino group.
12. The method according to claim 11 , wherein the carboxylic acid is selected from the group consisting of biotin, Biotin-Lys (Biotin), and Biotin-Cys (Biotin).
13. The method according to claim 11 , wherein the anhydride having thiol group is 3,3-dithiopropionic anhydride.
14. The method according to claim 11 , wherein the chloroformate is selected from the group consisting of a fluorescein chloroformate, a thiol chloroformate, and a cholesterol chloroformate.
15. The method according to claim 11 , wherein the isocyanate is selected from the group consisting of fluorescein isocyanates, thiol isocyanates, and cholesterol isocyanates.
16. The method according to claim 11 , wherein the chelate is selected from the group consisting of 1,4,7,10-Tetraazacyclododecane-1,4,7-tris-tert-butyl acetate-10-acetic acid, 1,4,7,10-tetrazadodecane-N,N′,N″,N′″-tetraacetic acid (DTPA), Diethylenetriamine-N,N,N″,N″-tetra-tert-butyl acetate-N′-acetic acid, DTPA-tetra(t-Bu ester), 1,4,7,10-Tetraazacyclododecane-1,4,7-tris-tert-butylacetate-10-(N-a-Fmoc-N-e-acetamido-L-lysine), and Fmoc-L-Lys-mono-amide-DOTA-tris(t-Bu ester).