Oligonucleotide production method
The invention provides a novel method for producing an oligonucleotide using a nucleoside or oligonucleotide that is easy to isolate and has high storage stability. The oligonucleotide production method includes a step of subjecting a nucleoside or oligonucleotide having a pseudo solid phase-protecting group in at least one location selected from the group consisting of 2′-position, 3′-position, 5′-position and a nucleobase moiety and having a 5′-hydroxyl group or a 3′-hydroxyl group, to H-phosphonation to convert the 5′-hydroxyl group or the 3′-hydroxyl group into an H-phosphonated form.
1. A production method of an oligonucleotide comprising at least one elongation reaction cycle, wherein the elongation reaction cycle comprises
a first step of deprotecting a first nucleoside or first oligonucleotide having a pseudo solid phase-protecting group in at least one location selected from the group consisting of 2′-position, 3′-position and a nucleobase moiety, having a 3′-hydroxyl group protected with a basic protecting group or a pseudo solid phase-protecting group, and having a 5′-hydroxyl group protected with a temporary protecting group, to remove the temporary protecting group to form a 5′-hydroxyl group,
a second step of converting the resultant 5′-hydroxyl group into an H-phosphonated form using an H-phosphonate reagent, wherein the H-phosphonated form is of the formula
a third step of forming an oligomer of the first nucleoside or first oligonucleotide with a second nucleoside or second oligonucleotide having a 3′-hydroxyl group and having a 5′-hydroxyl group protected with a temporary protecting group, by forming a phosphite diester bond from the 5′-hydroxyl group, now converted to the H-phosphonated form, of the first nucleoside or first oligonucleotide and the 3′-hydroxyl group of the second nucleoside or second oligonucleotide,
a fourth step of converting the phosphite diester bond of the oligomer into a phosphodiester bond, a thiophosphodiester bond, a boranophosphodiester bond, an aminophosphodiester bond, a phosphodiester bond protected with a basic protecting group, or a thiophosphodiester bond protected with a basic protecting group, and
a fifth step of adding a polar solvent to a reaction mixture obtained from at least one step selected from the group consisting of the first step to the fourth step to form a precipitate, and collecting the precipitate by solid liquid separation,
wherein the pseudo solid phase-protecting group is of formula (I):
wherein
* indicates a bond to a group protected by the pseudo solid phase-protecting group,
R 1 is a C10-30 alkyl group or a C10-30 alkenyl group,
s is an integer of 1 to 5,
L 1 is a single bond, a C1-6 alkylene group, a C2-6 alkenylene group or a C2-6 alkynylene group,
L 2 is a single bond, —COO—, —CON(R 2 )— (wherein R 2 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group), —OCO, —N(R 2 )CO— (wherein R 2 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group), —C(O)— or —O—,
L 3 is a single bond, a C1-6 alkylene group, a C2-6 alkenylene group or a C2-6 alkynylene group,
L 4 is a single bond, —COO—, —CON(R 2 )— (wherein R 2 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group), —OCO—, —N(R 2 )CO— (wherein R 2 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group), —C(O)— or —O—,
L 5 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group,
L 6 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group,
m is 0 or 1, and
when the pseudo solid phase-protecting group is present in the nucleobase moiety and when m in the pseudo solid phase-protecting group is 0, the number of single bonds represented by L 1 , L 2 , L 3 and L 4 in the pseudo solid phase-protecting group is 0 to 3.
2. The production method according to claim 1 , wherein the fourth step converts the phosphite diester bond of the oligomer into a phosphodiester bond, a thiophosphodiester bond, a boranophosphodiester bond or an aminophosphodiester bond.
3. The production method according to claim 1 , wherein the polar solvent is an alcohol solvent with 1 to 6 carbon atoms or a nitrile solvent with 1 to 6 carbon atoms.
4. The production method according to claim 1 , further comprising a sixth step of removing all of the basic protecting group, the temporary protecting group and the pseudo solid phase-protecting group.
5. The production method according to claim 1 wherein the first nucleoside or the first oligonucleotide has a hydroxyl group protected with a pseudo solid phase-protecting group at 3′-position.
6. The production method according to claim 1 , wherein the third step uses the second nucleoside.
7. The production method according to claim 1 , wherein the pseudo solid phase-protecting group is represented by the following formula (II):
wherein
* indicates a bond to a group protected by the pseudo solid phase-protecting group,
R 1 is a C10-30 alkyl group or a C10-30 alkenyl group,
s is an integer of 1 to 5,
L 1 is a single bond, a C1-6 alkylene group, a C2-6 alkenylene group or a C2-6 alkynylene group,
L 2 is a single bond, —COO—, —CON(R 2 )— (wherein R 2 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group), —OCO—, —N(R 2 )CO— (wherein R 2 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group) or —O—,
L 3 is a single bond, a C1-6 alkylene group, a C2-6 alkenylene group or a C2-6 alkynylene group,
L 4 is a single bond, —COO—, —CON(R 2 )— (wherein R 2 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group), —OCO—, —N(R 2 )CO— (wherein R 2 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group) or —O—, and
when the pseudo solid phase-protecting group is present in the nucleobase moiety, the number of single bonds represented by L 1 , L 2 , L 3 and L 4 in the pseudo solid phase-protecting group is 0 to 3.
8. The production method according to claim 1 , wherein the temporary protecting group is a tert-butyldimethylsilyl group, a 4,4′-dimethoxytrityl group or a levulinyl group.
9. The production method according to claim 1 , wherein the H-phosphonation step uses at least one H-phosphonate reagent selected from the group consisting of phosphorous acid, diphenyl phosphite, phenyl-H-phosphonate triethylammonium salt, p-toluyl-H-phosphonate triethylammonium salt, 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one and phosphorus trichloride.
10. The production method according to claim 1 , wherein the nucleobases present in the first to the fourth nucleosides and the first to the fourth oligonucleotides are each independently at least one selected from the group consisting of 6-aminopurin-9-yl group (adenine residue), 2-amino-6-hydroxypurin-9-yl group (guanine residue), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group (cytosine residue), 2-oxo-4-amino-5-methyl-1,2-dihydropyrimidin-1-yl group (5-methylcytosine residue), 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl group (uracil residue) and 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group (thymine residue).
11. The production method according to claim 1 , wherein the H-phosphonate reagent is selected from the group consisting of phosphorous acid, a diaryl phosphite, an aryl-H-phosphonate ammonium salt, and a phosphorus halide.
12. A production method of an oligonucleotide comprising at least one elongation reaction cycle, wherein the elongation reaction cycle comprises:
a first step of deprotecting a third nucleoside or third oligonucleotide having a pseudo solid phase-protecting group in at least one location selected from the group consisting of 2′-position, 5′-position and a nucleobase moiety, having a 5′-hydroxyl group protected with a basic protecting group or a pseudo solid phase-protecting group, and having a 3′-hydroxyl group protected with a temporary protecting group, to remove the temporary protecting group to form a 3′-hydroxyl group,
a second step of converting the resultant 3′-hydroxyl group into an H-phosphonated form using an H-phosphonate reagent, wherein the H-phosphonated form is of the formula
a third step of forming an oligomer of the third nucleoside or third oligonucleotide with a fourth nucleoside or fourth oligonucleotide having a 5′-hydroxyl group and having a 3′-hydroxyl group protected with a temporary protecting group, by forming a phosphite diester bond from the 3′-hydroxyl group, now converted to the H-phosphonated form, of the third nucleoside or third oligonucleotide and the 5′-hydroxyl group of the fourth nucleoside or fourth oligonucleotide,
a fourth step of converting the phosphite diester bond of the oligomer into a phosphodiester bond, a thiophosphodiester bond, a boranophosphodiester bond, or an aminophosphodiester bond, a phosphodiester bond protected with a basic protecting group, or a thiophosphodiester bond protected with a basic protecting group, and
a fifth step of adding a polar solvent to a reaction mixture obtained from any of the first step to the fourth step to form a precipitate, and collecting the precipitate by solid liquid separation,
wherein the pseudo solid phase-protecting group is of formula (I):
wherein
* indicates a bond to a group protected by the pseudo solid phase-protecting group,
R 1 is a C10-30 alkyl group or a C10-30 alkenyl group,
s is an integer of 1 to 5,
L 1 is a single bond, a C1-6 alkylene group, a C2-6 alkenylene group or a C2-6 alkynylene group,
L 2 is a single bond, —COO—, —CON(R 2 )— (wherein R 2 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group), —OCO—, —N(R 2 )CO— (wherein R 2 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group), —C(O)— or —O—,
L 3 is a single bond, a C1-6 alkylene group, a C2-6 alkenylene group or a C2-6 alkynylene group,
L 4 is a single bond, —COO—, —CON(R 2 )— (wherein R 2 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group), —OCO—, —N(R 2 )CO— (wherein R 2 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group), —C(O)— or —O—,
L 5 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group,
L 6 is a hydrogen atom, a C1-6 alkyl group, a C1-6 haloalkyl group, a C2-6 alkenyl group or a C2-6 haloalkenyl group,
m is 0 or 1, and
when the pseudo solid phase-protecting group is present in the nucleobase moiety and when m in the pseudo solid phase-protecting group is 0, the number of single bonds represented by L 1 , L 2 , L 3 and L 4 in the pseudo solid phase-protecting group is 0 to 3.
13. The production method according to claim 12 , wherein the fourth step converts the phosphite diester bond of the oligomer into a phosphodiester bond, a thiophosphodiester bond, a boranophosphodiester bond or an aminophosphodiester bond.
14. The production method according to claim 12 , wherein the polar solvent is an alcohol solvent with 1 to 6 carbon atoms or a nitrile solvent with 1 to 6 carbon atoms.
15. The production method according to claim 12 , further comprising a sixth step of removing all of the basic protecting group, the temporary protecting group and the pseudo solid phase-protecting group.
16. The production method according to claim 12 , wherein the third nucleoside or the third oligonucleotide has a hydroxyl group protected with a pseudo solid phase-protecting group at 5′-position.
17. The production method according to claim 12 , wherein the third step uses the fourth nucleoside.