Method for preparing ribonucleoside phosphorothioate
A method for preparing a phosphorothioate RNA based on the oxazaphospholidine method, wherein cyanoethoxymethyl group is used instead of tert-butyldimethylsilyl group as a protective group of 2′-hydroxyl group of RNA.
1. A method for preparing a ribonucleoside phosphorothioate represented by the following formula (I) or a salt thereof:
wherein, R 1 represents hydrogen atom or a protective group of hydroxyl group, Bs represents a nucleobase which may have a protective group, n represents 0 or an integer of 1 or larger, and n of X independently represents a divalent group represented by the following formula (II-Sp) or (II-Rp):
wherein, R 2 represents hydrogen atom or cyanoethoxymethyl group,
which comprises the step of condensing a compound represented by the following formula (III):
wherein, R 3 and R 4 independently represent a protective group of hydroxyl group, and one of R 3 and R 4 may represent a solid phase carrier bound via a linker as required, with an oxazaphospholidine ribonucleoside represented by the following formula (IVa) or (IVb):
wherein, CEM represents cyanoethoxymethyl group, R 5 represents a protective group of hydroxyl group, and R 6 represents an aryl group which may have a substituent, and then sulfurizing the resulting product.
2. The method according to claim 1 , wherein R 6 is a phenyl group.
3. The method according to claim 1 , wherein R 5 is a 4,4′-dimethoxytrityl group.
4. The method according to claim 1 , wherein the condensation is performed in the presence of an activator.
5. The method according to claim 4 , wherein N-(cyanomethyl)pyrrolidinium triflate or N-phenylimidazolium triflate is used as the activator.
6. The method according to claim 1 , wherein dimethyl thiuram disulfide is used as a sulfurizing agent.
7. The method according to claim 1 , which comprises the step of repeating the aforementioned step n+1 times using a compound represented by the formula (III) in which n is 0 as a starting material.
8. The method according to claim 1 , wherein the reaction is performed by the solid phase method.
9. The method according to claim 8 , wherein a compound represented by the formula (III), in which n is 0 and which is bound to a solid phase carrier optionally via a linker, is used.
10. The method according to claim 1 , wherein all of n+1 of X are divalent groups represented by the formula (II-Sp), or all of them are divalent groups represented by the formula (II-Rp).
11. A ribonucleoside phosphorothioate represented by the formula (I) mentioned in claim 1 , wherein, R 1 , Bs, n, and X have the same meanings as those defined above, and R 2 represents a cyanoethoxymethyl group, or a salt thereof.
12. An oxazaphospholidine ribonucleoside represented by the general formula (IVa) or (IVb) mentioned in claim 1 .