Method for preparing 3′-O-amino-2′-deoxyribonucleoside-5′-triphosphate

1. A method for preparing 3′-O-amino-2′-deoxyribonucleoside-5′-triphosphate, said method comprising the steps of:

(a) protecting 5′-hydroxy group of a 2′-deoxyribonucleoside;

(b) converting (S)-3′-hydroxy group of the compound obtained in step (a) into (R)-3′-hydroxy group;

(c) grafting the compound obtained in step (b) on a solid support functionalized with at least one N-hydroxyphthalimide moiety, wherein said at least one N-hydroxyphthalimide moiety is represented by formula (I):

wherein L represents a linker, under conditions allowing (R)-3′-hydroxy group of the compound obtained in step (b) to be substituted by a N-hydroxyphthalimide moiety of the functionalized solid support;

(d) deprotecting 5′-hydroxy group of the compound obtained in step (c);

(e) triphosphorylating 5′-hydroxy group of the compound obtained in step (d);

(f) reacting the compound obtained in step (e) with a cleaving reagent selected from the group consisting of primary amines, hydrazine and hydroxides; and,

(g) optionally recovering 3′-O-amino-2′-deoxyribonucleoside-5′-triphosphate obtained in step (f).

2. The method according to claim 1 , wherein said 2′-deoxyribonucleoside is selected from the group consisting of 2′-deoxythymidine, 2′-deoxyuridine, 2′-deoxycytidine, 2′-deoxyadenosine, 2′-deoxyguanosine, and a protected-base derivative thereof.

3. The method according to claim 1 , wherein step (α) comprises reacting a 2′-deoxyribonucleoside with trityl chloride, monomethoxytrityl chloride or dimethoxytrityl chloride.

4. The method according to claim 1 , wherein step (b) comprises the following sub-steps:

(b1′) converting (S)-3′-hydroxy group of the compound obtained in step (a) into (S)-3′-sulfonate group; and

(b2′) reacting the compound obtained in step (b1) with a hydroxide source.

5. The method according to claim 1 , wherein step (b) is carried out in the absence of phosphorous compound.

6. The method according to claim 1 , wherein step (c) is carried out in the presence of a phosphorous compound, and of an azo compound.

7. The method according to claim 1 , wherein step (c) comprises the following substeps:

(c1) converting (R)-3′-hydroxy group of the compound obtained in step (b) into (R)-3′-sulfonate group; and

(c2) reacting the compound obtained in step (c1) with a N-hydroxyphthalimide-functionalized solid support of formula (I).

8. The method according to claim 1 , wherein step (e) comprises the following sub-steps:

(e1) reacting the compound obtained in step (d) with 2-chloro-1,3,2-benzodioxaphosphorin-4-one or phosphoryl chloride; and

(e2) reacting the compound obtained in step (e1) with a pyrophosphate salt.

9. The method according to claim 1 , wherein said cleaving reagent in step (f) is selected from the group consisting of hydrazine, lithium hydroxide, cesium hydroxide, sodium hydroxide, potassium hydroxide, ammonium hydroxide, and R′—NH 2 , wherein R′ represents a (C 1 -C 6 )alkyl, a (C 2 -C 6 )alkenyl a (C 2 -C 6 )alkynyl, a (C 3 -C 12 )cycloalkyl, or a (C 6 -C 14 )aryl.

10. The method according to claim 1 , wherein said N-hydroxyphthalimide-functionalized solid support is made of a material selected from the group consisting of a polystyrene resin, silica, tentagel, glass, and a mixture thereof.

11. The method according to claim 1 , wherein L is —CH 2 —NH—(CO)—.

12. A 3′-O-amino-2′-deoxyribonucleoside-5′-triphosphate grafted on a solid support functionalized with at least one N-hydroxyphthalimide moiety, wherein said at least one N-hydroxyphthalimide moiety is represented by formula (I):

wherein L represents a linker.

13. The 3′-O-amino-2′-deoxyribonucleoside-5′-triphosphate of claim 12 , wherein L is —CH 2 —NH—C(O)—.