Reagents utilizing a serinol scaffold for labeling synthetic oligonucleotides
View Patent ↗Novel CE-phosphoramidites and CPG reagents have been synthesized from a serinol backbone. These reagents are useful to introduce functional groups or directly label oligonucleotides. The versatile serinol scaffold allows for labeling at any position (5′ or 3′ termini, or any internal position) during automated DNA synthesis. Multiple labels or functional groups can be achieved by repetitive coupling cycles. Optimal spacer arms and protected label moieties have been specially designed. Further, the natural 3-carbon atom internucleotide phosphate distance is retained when inserted internally.
1. A reagent having the following structure:
wherein:
R 1 =dimethoxytrityl (DMT), monomethoxytrityl (MMT), or other hydroxyl protecting group stable to oligonucleotide synthesis conditions;
R 2 =phosphoramidityl
wherein R 3 and R 4 are independently selected from the group consisting of C 1-10 branched alkyl, C 1-12 alkyl, and cyclic hydrocarbyls; and R 5 is a phosphite-protecting group
X 1 =—CO—CH 2 CH 2 or —CO—CH 2 ,
X 2 =—NH—, —S—, or —O—;
R 6 =a reporter substituent moiety selected from the group consisting of biotinyl, carboxyfluoresceinyl, propargyl, and a hapten-derived substituent moiety; or a suitable protecting group for X 2 .
2. The reagent according to claim 1 wherein R 3 ═R 4 =—CH(CH 3 ) 2 and R 5 =—O—(CH 2 ) 2 —CN.
3. The reagent according to claim 2 , having the following structure (Compound 5):
4. The reagent according to claim 2 , having the following structure (Compound 6):
5. The reagent according to claim 2 , having the following structure (Compound 7):
6. The reagent according to claim 2 , having the following structure (Compound 20):
7. A process to label or modify a synthetic oligonucleotide at any nucleoside position wherein the process comprises incorporating a reagent having the structure according to claim 1 into said oligonucleotide by phosphoramidite coupling during automated oligonucleotide synthesis.
8. A process to label or modify a synthetic oligonucleotide at any nucleoside position wherein the process comprises incorporating a reagent having the structure according to claim 2 into said oligonucleotide by phosphoramidite coupling during automated oligonucleotide synthesis.
9. A process to label or modify a synthetic oligonucleotide at any nucleoside position wherein the process comprises incorporating a reagent having the structure according to claim 3 into said oligonucleotide by phosphoramidite coupling during automated oligonucleotide synthesis.
10. A process to label or modify a synthetic oligonucleotide at any nucleoside position wherein the process comprises incorporating a reagent having the structure according to claim 4 into said oligonucleotide by phosphoramidite coupling during automated oligonucleotide synthesis.
11. A process to label or modify a synthetic oligonucleotide at any nucleoside position wherein the process comprises incorporating a reagent having the structure according to claim 5 into said oligonucleotide by phosphoramidite coupling during automated oligonucleotide synthesis.
12. A process to label or modify a synthetic oligonucleotide at any nucleoside position wherein the process comprises incorporating a reagent having the structure according to claim 6 into said oligonucleotide by phosphoramidite coupling during automated oligonucleotide synthesis.
13. A reagent having the following structure:
wherein:
R 1 =dimethoxytrityl (DMT), monomethoxytrityl (MMT), or other hydroxyl protecting group stable to oligonucleotide synthesis conditions;
R 2 is a long chain alkyl amine controlled pore glass attached through a succinyl linkage (-succinyl-CPG);
X 1 =—CO—CH 2 CH 2 or —CO—CH 2 ;
X 2 =—NH—, —S—, or —O—;
R 6 =a reporter substituent moiety selected from the group consisting of biotinyl, carboxyfluoresceinyl, propargyl, and a hapten-derived substituent moiety; or a suitable protecting group for X 2 .
14. The reagent according to claim 13 , having the following structure (Compound 8):
15. The reagent according to claim 13 , having the following structure (Compound 9):
16. The reagent according to claim 13 , having the following structure (Compound 10):
17. The reagent according to claim 13 , having the following structure (Compound 12):
18. The reagent according to claim 13 , having the following structure (Compound 21):
19. A process to label or modify a synthetic oligonucleotide at the 3′ terminal position by substituting the reagent having the structure according to claim 14 into said oligonucleotide for a standard nucleosidic solid support in automated oligonucleotide synthesis.
20. A process to label or modify a synthetic oligonucleotide at the 3′ terminal position by substituting the reagent having the structure according to claim 15 into said oligonucleotide for a standard nucleosidic solid support in automated oligonucleotide synthesis.
21. A process to label or modify a synthetic oligonucleotide at the 3′ terminal position by substituting the reagent having the structure according to claim 16 into said oligonucleotide for a standard nucleosidic solid support in automated oligonucleotide synthesis.
22. A process to label or modify a synthetic oligonucleotide at the 3′ terminal position by substituting the reagent having the structure according to claim 17 into said oligonucleotide for a standard nucleosidic solid support in automated oligonucleotide synthesis.
23. A process to label or modify a synthetic oligonucleotide at the 3′ terminal position by substituting the reagent having the structure according to claim 18 into said oligonucleotide for a standard nucleosidic solid support in automated oligonucleotide synthesis.
24. A process to label or modify a synthetic oligonucleotide at the 3′ terminal position by substituting the reagent having the structure according to claim 13 into said oligonucleotide for a standard nucleosidic solid support in automated oligonucleotide synthesis.