Processes and reagents for sulfurization of oligonucleotides
View Patent ↗The present invention relates to processes and reagents for oligonucleotide synthesis and purification. One aspect of the present invention relates to compounds useful for activating phosphoramidites in oligonucleotide synthesis. Another aspect of the present invention relates to a method of preparing oligonucleotides via the phosphoramidite method using an activator of the invention. Another aspect of the present invention relates to sulfur-transfer agents. In a preferred embodiment, the sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the present invention relates to a method of preparing a phosphorothioate by treating a phosphite with a sulfur-transfer reagent of the invention. In a preferred embodiment, the sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the present invention relates to compounds that scavenge acrylonitrile produced during the deprotection of phosphate groups bearing ethylnitrile protecting groups. In a preferred embodiment, the acrylonitrile scavenger is a polymer-bound thiol. Another aspect of the present invention relates to agents used to oxidize a phosphite to a phosphate. In a preferred embodiment, the oxidizing agent is sodium chlorite, chloramine, or pyridine-N-oxide. Another aspect of the present invention relates to methods of purifying an oligonucleotide by annealing a first single-stranded oligonucleotide and second single-stranded oligonucleotide to form a double-stranded oligonucleotide; and subjecting the double-stranded oligonucleotide to chromatographic purification. In a preferred embodiment, the chromatographic purification is high-performance liquid chromatography.
1. A compound represented by formula E:
wherein
X is O;
R 1 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R 2 is —C(S)N(R 3 )R 4 , —C(S)N(R 3 ) 2 , or —C(S)OR 4 ;
R 3 is H or alkyl; and
R 4 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.
2. The compound of claim 1 , wherein R 2
is —C(S)N(R 3 )R 4 , or —C(S)N(R 3 ) 2 , and R3 is H.
3. The compound of claim 1 , wherein R 4 is alkyl or aryl.
4. The compound of claim 3 , wherein R 4 is ethyl.
5. The compound of claim 1 , wherein R 1 is H.
6. The compound of claim 1 , wherein R 2 is —C(S)OR 4 .
7. A process for preparing a compound, represented by formula E:
comprising the steps of:
admixing about 1 equivalent of chlorocarbonyl sulfenyl chloride, about 1 equivalent of a thiourea compound, and about 1 equivalent of triethylamine in a container cooled with an ice-bath at about 0° C. under an atmosphere of argon, stirring the resultant mixture for about 6 hours, filtering said mixture, concentrating said mixture to give a residue, and recystallizing said residue from dichloromethane-hexanes to give the compound,
wherein:
X is O;
R 1 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R 2 is —C(S)N(R 3 )R 4 , —C(S)N(R 3 ) 2 , or —C(S)OR 4 ;
R 3 is H or alkyl; and
R 4 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.
8. The process of claim 7 , wherein R 1 is H.
9. The process of claim 7 , wherein R 2
is —C(S)N(R 3 )R 4 , or —C(S)N(R 3 ) 2 , and R 3 is H.
10. The process of claim 7 , wherein R 2
is —C(S)OR 4 .
11. The process of claim 7 , wherein R 4 is alkyl or aryl.
12. The process of claim 11 , wherein R 4 is ethyl.
13. A method of sulfurizing a phosphorus-containing oligonucleotide comprising reacting a phosphorus-containing oligonucleotide with a compound represented by formula E:
to sulfurize the phosphorus-containing oligonucleotide and form one or more phosphorothioate linkages in the oligonucleotide,
wherein:
X is O;
R 1 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R 2 is —C(S)N(R 3 )R 4 , —C(S)N(R 3 ) 2 , or —C(S)OR 4 ;
R 3 is H or alkyl; and
R 4 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.
14. The method of claim 13 , wherein R 1 is H.
15. The method of claim 13 , wherein R 2
is —C(S)N(R 3 )R 4 , or —C(S)N(R 3 ) 2 , and R 3 is H.
16. The method of claim 13 , wherein R 2 is —C(S)OR 4 .
17. The method of claim 13 , wherein R 4 is alkyl or aryl.
18. The method of claim 17 , wherein R 4 is ethyl.