Pyridinium salts as activators in the synthesis of stereodefined oligonucleotides

Abstract

The present invention relates to a method for preparing stereodefined phosphorothioate oligonucleotides, especially locked stereodefined phosphorothioate oligonucleotides with a high yield, using pyridinium acidic salts as a coupling activator.

Claims

1. A method for the synthesis of stereodefined, sugar modified oligonucleotides comprising a step of coupling an oxazaphospholidine monomer of formula (1a) or (1b): ##STR00048## wherein Nuc is a nucleoside comprising a protected 5′-hydroxyl and is attached to the oxygen atom of Formula 1a or 1b via its 3′ position; R is selected from the groups consisting of nitro, halogen, cyano, silyl, sulfone, C.sub.1-4 alkyl, C.sub.1-4 alkylsulfone, C.sub.6-14 arylsulfone, C.sub.6-14 aryl, C.sub.5-14 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of the silyl, alkyl, aryl, heteroaryl moieties can be unsubstituted or substituted by one or more group(s) selected from the group consisting of: C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy or silyl substituted by one or more substituent selected from C.sub.1-4-alkyl and C.sub.6-14 aryl; R.sup.1 is selected from hydrogen or C.sub.1-4 alkyl; and R.sup.5, R.sup.6 and R.sup.9 are independently selected from the group consisting of hydrogen, C.sub.1-4 alkyl, C.sub.5-6 cycloalkyl, C.sub.6-14 aryl, C.sub.5-14 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of which alkyl, cycloalkyl, aryl or heteroaryl can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy: or two of R.sup.5, R.sup.6 or R.sup.9 together form a heterocyclic ring comprising 3-7 carbon atoms, together with the N atom to which R.sup.5 is attached, wherein said heterocyclic ring can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy, with a nucleoside immobilized on a solid support wherein the coupling is performed in the presence of a pyridinium acidic salt coupling activator, wherein the pyridinium acidic salt coupling activator is pyridinium hydrochloride or pyridinium hydrobromide.

2. The method of claim 1, wherein the pyridinium acidic salt coupling activator is pyridinium hydrochloride.

3. The method of claim 1, wherein the pyridinium acidic salt coupling activator is pyridinium hydrobromide.

4. The method of claim 1, wherein the pyridinium acidic salt coupling activator is in a solvent selected from acetonitrile, pyridine in acetonitrile, methylimidazole as well as their mixtures.

5. The method of claim 4, wherein the pyridinium acid salt coupling activator in acetonitrile.

6. The method of claim 1, wherein the stereodefined monomer of formula (1a) or (1b) is a oxazaphospholidine of formula (2a) or (2b): ##STR00049## wherein R, R.sup.1, R.sup.9 and Nuc are as according to formula (1a) or (1b).

7. The method of claim 1, wherein the sugar modification is selected from the group consisting of the following LNA sugar modifications: ##STR00050## ##STR00051## ##STR00052## ##STR00053## wherein B is a nucleobase and Z and Z* are independently nucleotides.

8. The method of claim 7, wherein the sugar modification is selected from the group consisting of: beta-D-oxy-LNA, (R)-6′-methyl-beta-D-oxy LNA, (S)-6′-methyl-beta-D-oxy-LNA ((S)-cET) and ENA.

9. The method of claim 1, wherein the sugar modification is MOE: ##STR00054## wherein B is a nucleobase and Z and Z* are independently nucleotides.

10. The method of claim 1, wherein the oxazaphospholidine monomer is of formula (3a) or (3b): ##STR00055## wherein Nuc is as defined herein, R.sup.1 is H or methyl.

11. The method of claim 1, wherein the oxazaphospholidine monomer is of formula (3a) or (3b): ##STR00056## wherein Nuc is as defined herein, R.sup.1 is H or methyl.

12. The method of claim 1, wherein the oxazaphospholidine monomer is of formula (10a) or (10b): ##STR00057## wherein Nuc is as defined herein, R.sup.1 is H or methyl.

13. A composition comprising a pyridinium acidic salt activator, a solvent and an oxazaphospholidine monomer of formula (1a) or (1b) as defined in claim 1.

14. A method for the synthesis of stereodefined, sugar modified oligonucleotides comprising a step of coupling an oxazaphospholidine monomer of formula (1a) or (1b): ##STR00058## wherein Nuc is a nucleoside comprising a protected 5′-hydroxyl and is attached to the oxygen atom of Formula 1a or 1b via its 3′ position; R is selected from the groups consisting of nitro, halogen, cyano, silyl, sulfone, C.sub.1-4 alkyl, C.sub.1-4 alkylsulfone, C.sub.6-14 arylsulfone, C.sub.6-14 aryl, C.sub.5-14 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of the silyl, alkyl, aryl, heteroaryl moieties can be unsubstituted or substituted by one or more group(s) selected from the group consisting of: C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4 alkoxy or silyl substituted by one or more substituent selected from C.sub.1-4-alkyl and C.sub.6-14 aryl; R.sup.1 is selected from hydrogen or C.sub.1-4 alkyl; and R.sup.5, R.sup.6 and R.sup.9 are independently selected from the group consisting of hydrogen, C.sub.1-4 alkyl, C.sub.5-6 cycloalkyl, C.sub.6-14 aryl, C.sub.5-14 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of which alkyl, cycloalkyl, aryl or heteroaryl can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy: or two of R.sup.5, R.sup.6 or R.sup.9 together form a heterocyclic ring comprising 3-7 carbon atoms, together with the N atom to which R.sup.5 is attached, wherein said heterocyclic ring can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy, with a nucleoside immobilized on a solid support wherein the coupling is performed in the presence of a pyridinium acidic salt coupling activator, wherein the pyridinium acidic salt coupling activator is pyridinium hydrochloride or pyridinium hydrobromide, wherein the pyridinium acidic salt coupling activator is pyridinium hydrobromide in a range from about 0.05 to about 0.50 M.

15. A method for the synthesis of stereodefined, sugar modified oligonucleotides comprising a step of coupling an oxazaphospholidine monomer of formula (1a) or (1b): ##STR00059## wherein Nuc is a nucleoside comprising a protected 5′-hydroxyl and is attached to the oxygen atom of Formula 1a or 1b via its 3′ position; R is selected from the groups consisting of nitro, halogen, cyano, silyl, sulfone, C.sub.1-4 alkyl, C.sub.1-4 alkylsulfone, C.sub.6-14 arylsulfone, C.sub.6-14 aryl, C.sub.5-14 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of the silyl, alkyl, aryl, heteroaryl moieties can be unsubstituted or substituted by one or more group(s) selected from the group consisting of: C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy or silyl substituted by one or more substituent selected from C.sub.1-4-alkyl and C.sub.6-14 aryl; R.sup.1 is selected from hydrogen or C.sub.1-4 alkyl; and R.sup.5, R.sup.6 and R.sup.9 are independently selected from the group consisting of hydrogen, C.sub.1-4 alkyl, C.sub.5-6 cycloalkyl, C.sub.6-14 aryl, C.sub.5-14 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of which alkyl, cycloalkyl, aryl or heteroaryl can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy: or two of R.sup.5, R.sup.6 or R.sup.9 together form a heterocyclic ring comprising 3-7 carbon atoms, together with the N atom to which R.sup.5 is attached, wherein said heterocyclic ring can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy, with a nucleoside immobilized on a solid support wherein the coupling is performed in the presence of a pyridinium acidic salt coupling activator, wherein the pyridinium acidic salt coupling activator is pyridinium hydrochloride or pyridinium hydrobromide, wherein the pyridinium acidic salt coupling activator is pyridinium hydrobromide at about 0.25 M.

16. A method for the synthesis of stereodefined, sugar modified oligonucleotides comprising a step of coupling an oxazaphospholidine monomer of formula (1a) or (1b): ##STR00060## wherein Nuc is a nucleoside comprising a protected 5′-hydroxyl and is attached to the oxygen atom of Formula 1a or 1b via its 3′ position; R is selected from the groups consisting of nitro, halogen, cyano, silyl, sulfone, C.sub.1-4 alkyl, C.sub.1-4 alkylsulfone, C.sub.6-14 arylsulfone, C.sub.6-14 aryl, C.sub.5-14 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of the silyl, alkyl, aryl, heteroaryl moieties can be unsubstituted or substituted by one or more group(s) selected from the group consisting of: C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy or silyl substituted by one or more substituent selected from C.sub.1-4-alkyl and C.sub.6-14 aryl; R.sup.1 is selected from hydrogen or C.sub.1-4 alkyl; and R.sup.5, R.sup.6 and R.sup.9 are independently selected from the group consisting of hydrogen, C.sub.1-4 alkyl, C.sub.5-6 cycloalkyl, C.sub.6-14 aryl, C.sub.5-14 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of which alkyl, cycloalkyl, aryl or heteroaryl can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy: or two of R.sup.5, R.sup.6 or R.sup.9 together form a heterocyclic ring comprising 3-7 carbon atoms, together with the N atom to which R.sup.5 is attached, wherein said heterocyclic ring can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy, with a nucleoside immobilized on a solid support wherein the coupling is performed in the presence of a pyridinium acidic salt coupling activator, wherein the pyridinium acidic salt coupling activator is pyridinium hydrochloride or pyridinium hydrobromide, wherein the pyridinium acidic salt coupling activator is pyridinium hydrochloride in a range from about 0.25 to about 1 M.

17. A method for the synthesis of stereodefined, sugar modified oligonucleotides comprising a step of coupling an oxazaphospholidine monomer of formula (1a) or (1b): ##STR00061## wherein Nuc is a nucleoside comprising a protected 5′-hydroxyl and is attached to the oxygen atom of Formula 1a or 1b via its 3′ position; R is selected from the groups consisting of nitro, halogen, cyano, silyl, sulfone, C.sub.1-4 alkyl, C.sub.1-4 alkylsulfone, C.sub.6-14 arylsulfone, C.sub.6-14 aryl, C.sub.5-14 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of the silyl, alkyl, aryl, heteroaryl moieties can be unsubstituted or substituted by one or more group(s) selected from the group consisting of: C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy or silyl substituted by one or more substituent selected from C.sub.1-4-alkyl and C.sub.6-14 aryl; R.sup.1 is selected from hydrogen or C.sub.1-4 alkyl; and R.sup.5, R.sup.6 and R.sup.9 are independently selected from the group consisting of hydrogen, C.sub.1-4 alkyl, C.sub.5-6 cycloalkyl, C.sub.6-14 aryl, C.sub.5-14 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of which alkyl, cycloalkyl, aryl or heteroaryl can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy: or two of R.sup.5, R.sup.6 or R.sup.9 together form a heterocyclic ring comprising 3-7 carbon atoms, together with the N atom to which R.sup.5 is attached, wherein said heterocyclic ring can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy, with a nucleoside immobilized on a solid support wherein the coupling is performed in the presence of a pyridinium acidic salt coupling activator, wherein the pyridinium acidic salt coupling activator is pyridinium hydrochloride or pyridinium hydrobromide, wherein the pyridinium acidic salt coupling activator is pyridinium hydrochloride at about 0.50 M.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 depicts a series of graphs, FIGS. 1A, 1B, 1C, 1D and 1E, based on an Ultra Performance Liquid Chromatography—mass spectrometry tandem analysis. The graphs exhibit the analysis of the crude material after coupling of a 9-mer fully phosphorothioated DNA T oligonucleotide with a L-DNA T monomer using various coupling activators and solvents to obtain a 10-mer oligonucleotide in a method according to the invention.

(2) FIG. 2 depicts a series of graphs, FIGS. 2A, 2B and 2C, based on an Ultra Performance Liquid Chromatography—mass spectrometry tandem analysis. The graphs exhibit the analysis of the crude material after coupling of a 15-mer fully phosphorothioated DNA T oligonucleotide with a L-DNA T monomer using various coupling activators and solvents to obtain a 16-mer oligonucleotide in a method according to the invention.

(3) FIG. 3 depicts a series of graphs, FIGS. 3A, 3B, 3C and 3D, based on an Ultra Performance Liquid Chromatography—mass spectrometry tandem analysis. The graphs exhibit the analysis of the crude material after coupling of a 15-mer fully phosphorothioated DNA T oligonucleotide with a L-DNA T monomer using various coupling activators and solvents to obtain a 16-mer oligonucleotide in a method according to the invention.

(4) FIG. 4 depicts a series of graphs, FIGS. 4A, 4B and 4C, based on an Ultra Performance Liquid Chromatography—mass spectrometry tandem analysis. The graphs exhibit the analysis of the crude material after coupling of a 15-mer fully phosphorothioated DNA T oligonucleotide with an LNA.sup.mC monomer using various coupling activators and solvents to obtain a 16-mer oligonucleotide in a method according to the invention.

(5) FIG. 5 depicts a series of graphs, FIGS. 5A and 5B, based on an Ultra Performance Liquid Chromatography—mass spectrometry tandem analysis. The graphs exhibit the analysis of the crude material after coupling of a 15-mer fully phosphorothioated DNA T oligonucleotide with a LNA.sup.mC monomer using various coupling activators and solvents to obtain a 16-mer oligonucleotide in a method according to the invention.

(6) FIG. 6 depicts a series of graphs, FIGS. 6A, 6B, 6C, 6D and 6E, based on an Ultra Performance Liquid Chromatography—mass spectrometry tandem analysis. The graphs exhibit the analysis of the crude material after coupling of a 15-mer fully phosphorothioated DNA T oligonucleotide with a LNA.sup.mC monomer using various coupling activators and solvents to obtain a 16-mer oligonucleotide in a method according to the invention.

(7) FIG. 7 depicts a graph of the theoretical coupling yields using various activators as per example and FIG. 1.

(8) FIG. 8 depicts the synthesis of oligonucleotides with a coupling step according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

(9) In a first aspect, the invention relates to method for the synthesis of stereodefined, sugar modified oligonucleotides comprising a step of coupling an oxazaphospholidine monomer of formula (1a) or (1b):

(10) ##STR00016##
wherein Nuc is a nucleoside comprising a protected 5′-hydroxyl and is attached to the oxygen atom of Formula 1a or 1b via its 3′ position;
R is selected from the groups consisting of nitro, halogen, cyano, silyl, sulfone, C.sub.1-4 alkyl, C.sub.1-4 alkylsulfone, C.sub.6-14 arylsulfone, C.sub.6-14 aryl, C.sub.5-14 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of the silyl, alkyl, aryl, heteroaryl moieties can be unsubstituted or substituted by one or more group(s) selected from the group consisting of: C.sub.1-4 alkyl. C.sub.6-14 aryl and C.sub.1-4, alkoxy;
R.sup.1 is selected from the groups consisting of hydrogen and C.sub.1-4 alkyl; and
R.sup.5, R.sup.6 and R.sup.9 are independently selected from the group consisting of hydrogen, C.sub.1-4 alkyl, C.sub.5-6 cycloalkyl, C.sub.6-14 aryl, C.sub.3-4 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of which alkyl, cycloalkyl, aryl or heteroaryl can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy
alternatively, two of R.sup.5, R.sup.6 or R.sup.9 together form a heterocyclic ring comprising 3-7 carbon atoms, together with the N atom to which R.sup.5 is attached wherein said heterocyclic ring can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4 alkoxy.
with a nucleoside immobilized on a solid support wherein the coupling is performed in the presence of a pyridinium acidic salt coupling activator.

(11) In another aspect, the invention relates to method for the synthesis of stereodefined, sugar modified oligonucleotides comprising a step of coupling an oxazaphospholidine monomer of formula (1a) or (1b):

(12) ##STR00017##
wherein Nuc is a nucleoside comprising a protected 5′-hydroxyl and is attached to the oxygen atom of Formula 1a or 1b via its 3′ position;
R is selected from the groups consisting of nitro, halogen, cyano, silyl, sulfone, C.sub.1-4 alkyl, C.sub.1-4 alkylsulfone, C.sub.6-14 arylsulfone, C.sub.6-14 aryl, C.sub.5-14 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of the silyl, alkyl, aryl, heteroaryl moieties can be unsubstituted or substituted by one or more group(s) selected from the group consisting of: silyl substituted by one or more C.sub.1-4-alkyl and/or C.sub.6-14 aryl, C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4 alkoxy;
R.sup.1 is selected from the groups consisting of hydrogen and C.sub.1-4 alkyl; and
R.sup.5, R.sup.6 and R.sup.9 are independently selected from the group consisting of hydrogen, C.sub.1-4 alkyl, C.sub.f cycloalkyl, C.sub.6-14 aryl, C.sub.3-14 heteroaryl comprising one, two or three heteroatoms independently selected from O, N or S, each of which alkyl, cycloalkyl, aryl or heteroaryl can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy
or two of R.sup.5, R.sup.6 or R.sup.9 together form a heterocyclic ring comprising 3-7 carbon atoms, together with the N atom to which R.sup.5 is attached wherein said heterocyclic ring can be substituted by one, two or three substituents selected from the group consisting of C.sub.1-4 alkyl, C.sub.6-14 aryl and C.sub.1-4, alkoxy, with a nucleoside immobilized on a solid support wherein the coupling is performed in the presence of a pyridinium acidic salt coupling activator.

(13) In an embodiment of the method according to the invention, the pyridinium acidic salt coupling activator is selected from the group consisting of pyridinium hydrochloride, pyridinium hydrobromide, pyridinium trifluroacetate, pyridinium triflate and pyridinium mesylate.

(14) In an embodiment of the method according to the invention, the pyridinium acidic salt coupling activator is pyridinium triflate at about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 M.

(15) In an embodiment of the method according to the invention, the pyridinium acidic salt coupling activator is pyridinium triflate at about 0.3 M.

(16) In an embodiment of the method according to the invention, the pyridinium acidic salt coupling activator is pyridinium triflate at about 1 M.

(17) In an embodiment of the method according to the invention the pyridinium acidic salt coupling activator is pyridinium hydrobromide in a range from about 0.05 to about 0.50 M, for example about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 045 or 0.50 M.

(18) In an embodiment of the method according to the invention, the pyridinium acidic salt coupling activator is pyridinium hydrobromide at about 0.25 M.

(19) In an embodiment of the method according to the invention, the pyridinium acidic salt coupling activator is pyridinium hydrochloride in a range from about 0.25 to about 1 M, for example, 0.25, 0.30, 0.35, 0.40, 045, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 or 1 M.

(20) In an embodiment of the method according to the invention, the pyridinium acidic salt coupling activator is pyridinium hydrochloride at about 0.50 M.

(21) In an embodiment of the method according to the invention, the pyridinium acidic salt coupling activator is in a solvent selected from acetonitrile, pyridinium in acetonitrile, methyl imidazole as well as their mixtures.

(22) In an embodiment of the method according to the invention, the pyridinium acid salt coupling activator is in acetonitrile.

(23) In an embodiment of the method according to the invention the method the oxazaphospholidine monomer of formulae (1a) and (1b) are:

(24) ##STR00018##
wherein R, R.sup.1, R.sup.9 and Nuc are defined herein for formula (1a) or (1b).

(25) In an embodiment of the method according to the invention, the sugar modification is selected from the group consisting of the following LNA sugar modifications:

(26) ##STR00019## ##STR00020## ##STR00021## ##STR00022## wherein B is a nucleobase and Z and Z* are independently nucleotides.

(27) In an embodiment of the method according to the invention the sugar modification is selected from the group consisting of: beta-D-oxy-LNA, 6′-methyl-beta-D-oxy LNA. (S)-6′-methyl-beta-D-oxy-LNA ((S)-cET) and ENA.

(28) In an embodiment of the method according to the invention, the sugar modification is MOE:

(29) ##STR00023## wherein B is a nucleobase and Z and Z* are independently nucleotides.

(30) In each embodiment of the method according to the invention described herein, the oxazaphospholidine monomer can be of formula (3a) or (3b):

(31) ##STR00024##
wherein Nuc is as defined herein, R.sup.1 is H or methyl.

(32) In an embodiment, the invention is method for the synthesis of stereodefined, oligonucleotides that comprise a ribose modification selected from the group consisting of beta-D-oxy-LNA, 6′-methyl-beta-D-oxy LNA, (S)-6′-methyl-beta-D-oxy-LNA ((S)-cET) and MOE, said method comprising a step of coupling an oxazaphospholidine monomer of formula (3a) or (3b):

(33) ##STR00025##
wherein Nuc is a nucleoside comprising a protected 5′-hydroxyl and is attached to the oxygen atom of Formula 1a or 1b via its 3′ position and R.sup.1 is selected from the groups consisting of hydrogen and C.sub.1-4 alkyl; with a nucleoside immobilized on a solid support wherein the coupling is performed in the presence of pyridinium hydrobromide at 0.25 M or pyridinium hydrochloride at 0.50 M as an activator in acetonitrile.

(34) In an embodiment, the invention is method for the synthesis of stereodefined, oligonucleotides that comprise a ribose modification selected from the group consisting of beta-D-oxy-LNA, (S)-6′-methyl-beta-D-oxy-LNA ((S)-cET) and MOE, said method comprising a step of coupling an oxazaphospholidine monomer of formula (3a) or (3b):

(35) ##STR00026##
wherein Nuc is a nucleoside comprising a protected 5%-hydroxyl and is attached to the oxygen atom of Formula 1a or 1b via its 3′ position and R.sup.1 is selected from the groups consisting of hydrogen and C.sub.1-4 alkyl; with a nucleoside immobilized on a solid support wherein the coupling is performed in the presence of pyridinium hydrobromide at 0.25 M or pyridinium hydrochloride at 0.50 M as an activator in acetonitrile.

(36) In an embodiment, the invention is method for the synthesis of stereodefined, oligonucleotides that comprise a ribose modification selected from the group consisting of beta-D-oxy-LNA, (S)-6′-methyl-beta-D-oxy-LNA ((S)-cET) and MOE, said method comprising a step of coupling an oxazaphospholidine monomer of formula (3a) or (3b):

(37) ##STR00027##
wherein Nuc is a nucleoside comprising a protected 5′-hydroxyl and is attached to the oxygen atom of Formula 1a or 1b via its 3‘ position and R’ is selected from the groups consisting of hydrogen and C.sub.1-4 alkyl; with a nucleoside immobilized on a solid support wherein the coupling is performed in the presence of pyridinium hydrobromide at 0.25 M as an activator in acetonitrile.

(38) In each embodiment described herein, the oxazaphospholidine monomer can be of formula (4a) or (4b):

(39) ##STR00028##
wherein B is a nucleobase, PG is an hydroxyl protecting group and R.sup.1 is selected from the groups consisting of hydrogen and C.sub.1-4 alkyl.

(40) In some or all embodiments, the nucleoside immobilized on a solid support can be of formula (5):

(41) ##STR00029##
wherein B′ is a nucleobase.

(42) In an embodiment, the invention is method for the synthesis of stereodefined, oligonucleotides that comprises the step of coupling an oxazaphospholidine monomer of formula (4a) or (4b):

(43) ##STR00030##
wherein R.sup.1 is selected from the groups consisting of hydrogen and C.sub.1-4 alkyl and B is a nucleobase, with a nucleoside immobilized on a solid support of formula:

(44) ##STR00031##
wherein B′ is a nucleobase and the coupling is performed in the presence of pyridinium hydrobromide at 0.25 M as an activator in acetonitrile.

(45) Another aspect of the invention is a composition comprising a pyridinium acidic salt activator, a solvent and a oxazaphospholidine of formula (1a) or (1b), wherein the pyridinium acidic salt activator, the solvent and the oxazaphospholidine of formula (1a) or (1b) are as described herein.

(46) In an embodiment, the composition of the invention comprises pyridinium hydrochloride activator at 0.50 M in acetonitrile and an oxazaphospholidine of formula (3a) or (3b):

(47) ##STR00032##
wherein R.sup.1 is selected from hydrogen or C.sub.1-4 alkyl.

(48) In an embodiment, the composition of the invention comprises pyridinium hydrobromide activator at about 0.25 M in acetonitrile and a compound of formula (3a) or (3b):

(49) ##STR00033##
wherein R.sup.1 is selected from hydrogen or C.sub.1-4 alkyl.

(50) Another aspect of the invention is the use of a pyridinium acidic salt activator for the coupling of an oxazaphospholidine monomer of formula (1a) or (1b) as defined herein with a nucleoside immobilized on a solid support.

(51) In an embodiment, a pyridinium hydrochloride activator is used at 0.50 M in acetonitrile for coupling of a nucleoside immobilized on a solid support with an oxazaphospholidine monomer of formula (3a) or (3b):

(52) ##STR00034##
wherein R.sup.1 is selected from hydrogen or C.sub.1-4 alkyl.

(53) In an embodiment, a pyridinium hydrobromide activator is used at about 0.25 M in acetonitrile for coupling of a nucleoside immobilized on a solid support with an oxazaphospholidine monomer of formula (3a) or (3b):

(54) ##STR00035##
wherein R.sup.1 is selected from hydrogen or C.sub.1-4 alkyl.

(55) Another aspect of the invention is an oligonucleotide manufactured according to the method of the invention.

(56) In some embodiments B or B′ is a nucleobase selected from the group consisting of adenine, guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.

(57) In some embodiments B or B′ is a purine nucleobase. In some embodiments B or B′ is a pyrimidine nucleobase. In some embodiments B or B′ is adenine. In some embodiments, B or B′ is thymidine. In some embodiments, B or B′ is guanine. In some embodiments, B or B′ is cytosine. In some embodiments, when B or B′ is cytosine, B is 5-methyl-cytosine.

(58) In some embodiments, B or B′ is other than cytosine, for example, when the monomer is a D-DNA monomer, e.g. of formula 20 or 22. In some embodiments, e.g. when the monomer is a D-DNA-C, B is other than acetyl (Ac) protected cytosine.

(59) It should be understood that for use in oligonucleotide synthesis the nucleobase group B or B′ may be protected in the amidite monomers (thymidine is often used without a protection group). Suitable protection groups include dimethyformamide (DMF), dimethoxytrityl (DMT) or an acyl protection group, such as isobutyryl (iBu), or an acetyl protection group (Ac) or a benzoyl protection group (Bz).

(60) In some embodiments, e.g. when the monomer is an L-LNA-G, B is other than DMF protected guanine (G). R.sup.3=is selected from the group consisting of CH.sub.2ODMTr, CH.sub.2-Alkyl-O-DMTr, CH-Me-O-DMTr, CH.sub.2OMMTr, CH.sub.2-Alkyl-O-MMTr, CH(Me)-O-MMTr, CH—R.sup.a—O-DMTrR.sup.b, and CH—R.sup.a—O-MMTrR.sup.b;

(61) R.sup.2 is selected from the groups consisting of halo, such as —F, amino, azido, —SH, —CN, —OCN, —CF.sub.3, —OCF.sub.3, —O(R.sup.m)-alkyl, —S(R.sup.m)-alkyl, —N(R.sup.m)-alkyl, —O(R.sup.m)-alkenyl, —S(R.sup.m)-alkenyl, —N(R.sup.m)-alkenyl: —O(R.sup.m)-alkynyl, —S(R.sup.m)-alkynyl or —N(R.sup.m)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH.sub.2).sub.2SCH.sub.2, O—(CH.sub.2).sub.2—O—N(R.sup.m)(R.sup.n) or O—CH.sub.2C(═O)—N(R.sup.m)(R.sup.n), —O—(CH.sub.2).sub.2OCH.sub.3, and —O—CH.sub.3, where each R.sup.m and R.sup.n are independently, H, an amino protecting group or substituted or unsubstituted C.sub.1-10 alkyl;

(62) R.sup.4 is selected from the group consisting of alkyl, cycloalkyl, cycloheteroalkyl, O-alkyl, S-alkyl, NH-alkyl, and hydrogen; In some embodiments, R.sup.4 is hydrogen. In some embodiments, R.sup.4 is hydrogen, and R.sup.2 is selected from the group consisting of —O—CH.sub.3, and —O—(CH.sub.2OCH.sub.3.

(63) Or in some embodiments, R.sup.2 and R.sup.4 together designate a bivalent bridge, such as consisting of 1, 2, 3 groups/atoms selected from the group consisting of —C(R.sup.aR.sup.b)—, —C(R.sup.a)═C(R.sup.b), —C(R.sup.a)═N, O, —Si(R.sup.a).sub.2—, S—, —SO.sub.2—, —N(R.sup.a)—, and >C═Z;

(64) wherein R.sup.a and, when present R.sup.b, each is independently selected from hydrogen, optionally substituted C.sub.1-6-alkyl, optionally substituted C.sub.2-6-alkenyl, optionally substituted C.sub.2-6-alkynyl, hydroxy, optionally substituted C.sub.1-6-alkoxy, C.sub.2-6-alkoxyalkyl, C.sub.2-6-alkenyloxy, carboxy, C.sub.1-6-alkoxycarbonyl, C.sub.1-6-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, hetero-aryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C.sub.1-6-alkyl)amino, carbamoyl, mono- and di(C.sub.1-6-alkyl)-amino-carbonyl, amino-C.sub.1-6-alkyl-aminocarbonyl, mono- and di(C.sub.1-6-alkyl)amino-C.sub.1-6-alkyl-aminocarbonyl, C.sub.1-6-alkyl-carbonylamino, carbamido, C.sub.1-6-alkanoyloxy, sulphono, C.sub.1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C.sub.1-6-alkylthio, halogen, where aryl and heteroaryl may be optionally substituted and where two geminal substituents R.sup.a and R.sup.b together may designate optionally substituted methylene (═CH.sub.2), wherein for all chiral centers, asymmetric groups may be found in either R or S orientation.

(65) In some embodiments, when incorporated into an oligonucleotide, the nucleoside (Z) confers a higher binding affinity to a complementary RNA target than an equivalent DNA nucleoside. Such nucleosides are referred to as high affinity nucleosides. Examples of high affinity nucleosides include 2′-O-MOE, 2′-fluoro, 2′-O-methyl, and LNA nucleosides. In the embodiments, where the nucleoside is a high affinity nucleoside R.sup.3 may, for example, be CH.sub.2—O-DMTr or CH.sub.2—O-MMTr.

(66) In some embodiments. R.sup.2 is selected from the group consisting of fluoro (—F). —O—(CH.sub.2).sub.2OCH.sub.3, and —O—C.sub.1-3 alkyl, such as —O—CH.sub.3. In such embodiments, optionally R.sup.4 is hydrogen.

(67) In some embodiments, the nucleoside is a LNA nucleoside (also known as a bicyclic nucleoside) comprising a 2′-4′ bridge (biradicle).

(68) In some embodiments, R.sup.2 and R.sup.4 together designate a bivalent bridge selected from the group consisting of bridge —C(R.sup.aR.sup.b)—O—. —C(R.sup.aR.sup.b) C(R.sup.aR.sup.b)—O—, —CH.sub.2—O—, —CH.sub.2CH.sub.2—O—, —CH(CH.sub.3)—O—. In some embodiments, R.sup.2 and R.sup.4 designate the bivalent bridge —CH.sub.2—O-(methylene-oxy also known as oxy-LNA) or —CH(CH.sub.3)—O— (methyl-methylene-oxy). The —CH(CH.sub.3)—O— bridge introduces a chiral center at the carbon atom within the bridge, in some embodiments this is in the S position (for example a nucleoside known in the art as (S)cET—see EP1984381)). In some embodiments, R.sup.2 and R.sup.4 designate the bivalent bridge —CH.sub.2—O— wherein the bridge is in the beta-D position (beta-D-oxy LNA). In some embodiments, R.sup.2 and R.sup.4 designate the bivalent bridge —CH.sub.2—O— wherein the bridge is in the alpha-L position (alpha-L-D-oxy LNA). In some embodiments, R.sup.2 and R.sup.4 designate the bivalent bridge —CH.sub.2—S— (thio LNA), or —CH.sub.2—NH.sub.2— (amino LNA). In the embodiments where R.sup.2 and R.sup.4 together designate a bivalent bridge, R.sup.3 may, for example be CH.sub.2—O-DMTr or CH.sub.2—O-MMTr.

(69) In some embodiments where the nucleoside (Nuc) is a bicyclic nucleotide (LNA) such as beta-D-oxy LNA, R is aryl, such as phenyl, and R.sup.1 is hydrogen or C.sub.1-3 alkyl. In such am embodiment, R.sup.5 and R.sup.6 may together form a heterocyclic ring, such as a five membered heterocyclic ring, as described herein (e.g. see formula 2a and 2b).

(70) In some embodiments, the compound of formula (1a) or (1b) is selected from the group consisting of formula 4a, 4b, 5a, 5b, 6a, 6b, 7a and 7b.

(71) ##STR00036## ##STR00037##

(72) In some embodiments, the compound of formula (1a or 1b) is selected from the group consisting of formula 8a, 8b, 8c or 8d; or 9a, 9b, 9c or 9d:

(73) ##STR00038##

(74) In some embodiments, the compound of formula (1a or 1b) is selected from the group consisting of formula of formula (10a) or (10b):

(75) ##STR00039##

(76) In some embodiments, the nucleobase B is adenine, such as Bz protected adenine. In some embodiments, the nucleobase B is thymine. In some embodiments, the monomer is a D-DNA-A monomer (e.g. the monomer is of formula 9c and the nucleobase B is adenine, such as Bz protected adenine). The examples illustrate that D-DNA-A monomers (e.g. of formula 9c), L-LNA-A monomers and L-LNA-T monomers (e.g. of formula 8a or 8b) show improved coupling when used in acetonitrile/aromatic heterocyclic solvents, as according to the invention.

(77) DMF Protected L-LNA-G

(78) As illustrated in PCT/EP2017/060985, DMF protected L-LNA-G monomers are poorly soluble in acetonitrile solvents. An L-LNA monomer can be defined either by the stereochemistry of chiral auxiliary of the monomer, or the stereochemistry of the internucleoside linkage which the monomer forms when it is incorporated into an oligonucleotide (the two features are structurally linked, and L monomer results in the creation of a Sp phosphorothioate linkage). An L-LNA monomer is represented by formula 3a, wherein in R.sup.4 and R.sup.2 form R.sup.2 and R.sup.4 together designate a bivalent bridge. See for example the monomers of formula 4a, 5a, 8a and 8b.

(79) In some embodiments, the oxazaphospholidine monomer is not an L-LNA monomer comprising a DMF protected guanine nucleobase.

(80) In some embodiments the DMF protected guanine group (B) has the following structure:

(81) ##STR00040##

(82) In some embodiments, the oxazaphospholidine monomer is not a monomer of formula 11 or 12:

(83) ##STR00041##

(84) wherein R, R.sup.1, R.sup.3, R.sup.5, R.sup.6 & R.sup.9 are as according to the monomer of formula 1, and wherein for the monomer of formula 11, X and Y together designate a bivalent bridge (e.g. as per R.sup.2 and R.sup.4 herein, such as a bridge selected from the group consisting of bridge —C(R.sup.aR.sup.b)—O—, —C(R.sup.aR.sup.b)C(R.sup.aR.sup.b)—O—, —CH.sub.2—O—, —CH.sub.2CH.sub.2—O—, —CH(CH.sub.3)—O—. In some embodiments, X and Y designate the bivalent bridge —CH.sub.2—O— (methylene-oxy also known as oxy-LNA) or —CH(CH.sub.3)—O— (methyl-methylene-oxy). The —CH(CH.sub.3)—O— bridge introduces a chiral center at the carbon atom within the bridge, in some embodiments this is in the S position (for example a nucleoside known in the art as (S)cET—see EP1984381)). In some embodiments, X and Y designate the bivalent bridge —CH.sub.2—O— wherein the bridge is in the beta-D position (beta-D-oxy LNA). In some embodiments, X and Y designate the bivalent bridge —CH.sub.2—O— wherein the bridge is in the alpha-L position (alpha-L-D-oxy LNA). In some embodiments, X and Y designate the bivalent bridge —CH.sub.2—S— (thio LNA), or —CH.sub.2—NH.sub.2— (amino LNA). In the embodiments where X and Y together designate a bivalent bridge, R.sup.3 may, for example be CH.sub.2—O-DMTr or CH.sub.2—O-MMTr.

(85) In some embodiments, the oxazaphospholidine monomer is a monomer of formula 13 or 14:

(86) ##STR00042##

(87) Wherein X, Y, R, R.sup.1, R.sup.9 and R.sup.3 are as per formula 11 and 12. The exocyclic oxygen of the guanine base may optionally be protected, e.g. with a cyano group.

(88) In some embodiments, the oxazaphospholidine monomer is a monomer of formula or 16:

(89) ##STR00043##

(90) Wherein X, Y, R.sup.1 and R.sup.3 are as per formula 11 and 12. The exocyclic oxygen of the guanine base may optionally be protected, e.g. with a cyano group. In some embodiments of formula 15 or 16, R.sup.1 is hydrogen. In some embodiments of formula 15 or 16, R.sup.3 is CH.sub.2—O-DMTr or CH.sub.2—O-MMTr. In some embodiments, the oxazaphospholidine monomer of the invention comprises an acyl-protected nucleoside (Z).

(91) Acyl Protected L-LNA-G

(92) As illustrated in the examples, DMF protected L-LNA-G monomers are poorly soluble in acetonitrile solvents. However, we have previously identified that the use of acyl protection groups on the guanine nucleoside of L-LNA-G monomers overcomes the solubility problem.

(93) In some embodiments, the oxazaphospholidine monomer is an L-LNA monomer comprising an acyl protected guanine nucleobase, such as an isobutyryl-protected guanine.

(94) In some embodiments, the oxazaphospholidine monomer is an L-LNA-G monomer of formula 23, 24, 25, 26, 27, 28, 29 or 30:

(95) ##STR00044## ##STR00045## ##STR00046##

(96) wherein, R, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.9 and R.sup.6 are as per the compound of the invention, and —C(═O)—R.sup.7 is the acyl protecting group on the exocyclic nitrogen of the guanine base, and R.sup.8 when present is a protecting group on the guanine exocyclic oxygen. In some embodiments R.sup.8 is cyanoethyl. In some embodiments, R is phenyl, R.sup.1 is hydrogen or methyl, and R.sup.3 is optionally CH.sub.2—O-DMTr or CH.sub.2—O-MMTr. In some embodiments, R.sup.7 is isobutyryl. In formula's 31 and 32, Y and X are as per formula 11.

(97) In some embodiments, the oxazaphospholidine monomer is selected from the group consisting of an L-LNA-T, D-DNA-A, D-DNA-C, L-LNA-C, and L-LNA-G (other than DMF protected L-LNA-G) or a L-DNA-C and L-DNA-T oxazaphospholidine monomer. As illustrated in the examples, these monomers show an improved coupling efficacy when used in the coupling solvent compositions of the invention, in addition to the solubility and stability benefits seen with in general for oxazaphospholidine monomers.

(98) Solvent Compositions (solutions)

(99) In some embodiments, the coupling step b) of the method of the invention uses an acetonitrile solution comprising an oxazaphospholidine monomer, acetonitrile and an aromatic heterocyclic solvent.

(100) In some embodiments, the acetonitrile solution further comprises an activator. Numerous activators for use in phosphoramidite oligonucleotide synthesis are known—they typically comprise acidic azole catalysts, such as 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, and 4,5-dicyanoimidazole. These activators are not necessarily useful in oxazaphospholidine synthesis.

(101) In some embodiments, the aromatic heterocyclic solvent has a pKa of about 4-about 7. In some embodiments, the aromatic heterocyclic solvent has a pKa of about 7-about 17 in water at 20° C.

(102) In some embodiments, the aromatic heterocyclic solvent is an aromatic heterocyclic base.

(103) In some embodiments, the aromatic heterocyclic solvent is an aromatic heterocyclic acid.

(104) In some embodiments, the aromatic heterocyclic solvent is selected from the group consisting of pyridine, 2-picoline, 4-picoline, 3-picoline, lutidine, and pyrrole.

(105) In some embodiments, the aromatic heterocyclic solvent is pyridine.

(106) In some embodiments, the aromatic heterocyclic solvent is pyrrole.

(107) In some embodiments, the aromatic heterocyclic solvent is 3-picoline.

(108) In some embodiments, the concentration (v/v), of aromatic heterocyclic solvent in acetonitrile is between about 0.1% and about 50% (v/v). In some embodiments, the concentration (v/v), of aromatic heterocyclic solvent in acetonitrile is between about 0.5% and about 40% (v/v). In some embodiments, the concentration (v/v), of aromatic heterocyclic solvent in acetonitrile is between about 0.5% and about 30% (v/v). In some embodiments, the concentration (v/v), of aromatic heterocyclic solvent in acetonitrile is between about 0.5% and about 25% (v/v). In some embodiments, the concentration (v/v), of aromatic heterocyclic solvent in acetonitrile is between about 0.5% and about 10% (v/v). In some embodiments, the concentration (v/v), of aromatic heterocyclic solvent in acetonitrile is between about 0.5% and about 5% (v/v). In some embodiments, the concentration (v/v), of aromatic heterocyclic solvent in acetonitrile is between about 1% and about 5% (v/v). In some embodiments, the concentration (v/v), of aromatic heterocyclic solvent in acetonitrile is between about 1% and about 4% (v/v). In some embodiments, the concentration (v/v), of aromatic heterocyclic solvent in acetonitrile is between about 0.5% (v/v) and about 10% (v/v), such as between about 1% (v/v) and about 5% (v/v), such as between about 2-3% (v/v), such as about 2.5% (v/v). In these embodiments, optionally the aromatic heterocyclic base solvent is pyridine.

(109) In some embodiments, wherein the aromatic heterocyclic solvent is pyridine, the concentration (v/v), of aromatic heterocyclic solvent in acetonitrile is between about 0.5% and about 10%, such as between about 1% and about 5%, such as between about 2-3%, such as about 2.5% or about 3.5%, or between about 2-4%.

(110) In some embodiments, wherein the aromatic heterocyclic solvent is pyrrole, the concentration (v/v), of aromatic heterocyclic solvent in acetonitrile is between about 0.5% and about 10%, such as between about 1% and about 5%, such as between 2-4% or about 2-3%, such as about 2.5%.

(111) In some embodiments, wherein the aromatic heterocyclic solvent is 3-picoline, the concentration (v/v), of aromatic heterocyclic solvent in acetonitrile is between about 0.5% and about 10%, such as between about 1% and about 5%, such as between 2-4%, or about 2-3%, such as about 2.5%.

(112) Pyridinium Acidic Salt Activators

(113) Activators are reagents used prior to or during the coupling step of oligonucleotide synthesis which activate the oxazaphospholidine monomer to allow coupling of the monomer to the 5′ terminal group attached to the solid support or oligonucleotide chain.

(114) In the method of the invention the activator is a pyridinium acidic salt. Pyridinium acidic salts can be selected from the group consisting of as pyridinium hydroiodide, pyridinium camphor sulphonic acid, pyridinium hydrochloride, pyridinium hydrobromide, pyridinium trifluroacetate, pyridinium triflate and pyridinium mesylate. Other pyridinium acidic salts can also be used

(115) Additional Activators

(116) Other activators can be added in addition, for example, in some embodiments, the additional activator is selected from the group consisting of CMPT (N-(Cyanomethyl)pyrrolidinium triflate (CMPT). N-(phenyl)imidazolium triflate (PhIMT), benzimidazolium triflate (BIT), 4,5-dicyanoimidazole (DCI), tetrazole, and 5-(Benzylthio)-1H-tetrazole.

(117) In some embodiments, the additional activator is 4,5-dicyanoimidazole (DCI).

(118) In some embodiments, the solvent composition comprises about 0.5-about 2M DCI (or the other activators of claim 13), such as about 1 M DCI (or the other activators of claim 13).

(119) In some embodiments, the solvent composition further comprises N-methylimidazole, such as N-methylimidazole in a concentration of 0.01-about 1 M N-methylimidazole, such as about 0.1M N-methylimidazole.

(120) In some embodiments, the activator comprises N-methylimidazole. In some embodiments, the activator comprises 4,5-dicyanoimidazole (DCI), tetrazole, or 5-(Benzylthio)-1H-tetrazole. In some embodiments, the activator comprises 4,5-dicyanoimidazole (DCI), tetrazole, or 5-(Benzylthio)-1H-tetrazole and N-methylimidazole.

(121) In some embodiments, the concentration of N-methylimidazole used is 0.01M-about 1M N-methylimidazole, such as about 0.1M N-methylimidazole. In some embodiments, the acetonitrile solution comprises N-methylimidazole in a concentration of 0.01M-about 1M N-methylimidazole, such as about 0.1M N-methylimidazole.

(122) In some embodiments, the activator is DCI or tetrazole, or 5-(Benzylthio)-1H-tetrazole, which may be used at a concentration (e.g. in the acetonitrile solution of the invention) of about 0.5-about 2M, such as about 1M.

(123) The stereodefined, sugar modified oligonucleotides that are synthesized according to the invention can be oligonucleotides that comprise 2′-sugar modified oligonucleosides. In other words, they can be 2′-sugar modified oligonucleotides.

(124) The stereodefined, sugar modified oligonucleotides that are synthesized according to the invention can be oligonucleotides that comprise Locked Nucleic Acid Nucleosides (LNA nucleosides). In other words, they can be Locked Nucleic Acid Nucleotides (LNA nucleotides).

(125) In some embodiments the activator is 4,5-dicyanoimidazole (DCI). In some embodiments, the solvent composition comprises about 0.5-about 2M DCI, such as about 1M DCI. It will be recognised that in order to optimise coupling efficacy, it may be necessary to optimize the amount of activator used, as is illustrated in the examples. In some embodiments the concentration of DCI activator uses is between 0.5M and 1M DCI. In some embodiments when the activator is DCI, the solvent composition further comprises N-methylimidazole (NMI), such as N-methylimidazole in a concentration of 0.01-about 1M N-methylimidazole, such as about 0.1M N-methylimidazole. NMI is an agent which can enhance the solubility of other activators such as DCI.

(126) The method of the invention provides a path to the synthesis of stereodefined oligonucleotides with a higher coupling efficiency than in the prior art as evidenced by the experiments conducted by the inventors. A non-limiting example of a generic process for preparing stereodefined oligonucleotides is depicted on FIG. 8. Referring to example 1 and FIG. 1 that summarize the experiments conducted by the inventors, it can be seen that the coupling of a 9-mer with a monomer to obtain a 10-mer gives a better yield with a pyridinium hydrochloride salts (94.7%) than with other non pyridinium acidic salt activators such as DCI+NMI, phenyl imidazolium triflate, benzimidazolium triflate+NMI or benzimidazolium triflate. Referring to FIG. 1B, the peaks of the resulting 10-mer and remaining 9-mer and by-product di-mer (18-mer) are more favourable than with the other coupling activators.

(127) The same is shown with example 2 and FIG. 2, in particular FIG. 1B.

(128) Examples 3, 4 and FIGS. 3, 4 also evidence the superiority of pyridinium acidic salt activators at various concentrations over DCI+NMI.

(129) Examples 5, 6 and FIGS. 5, 6 show that pyridinium hydrobromide is particularly efficient among others pyridinium acidic salt activators.

(130) FIG. 7 shows the theoretical overall reaction yields of oligomers as a function of different activators and lengths of oligonucleotides as per FIG. 6. This figure shows that small changes in coupling yields have dramatic effects on overall oligomer yield.

(131) The following non-limiting examples will further illustrate the invention.

EXAMPLES

Example 1

(132) To examine the effect of the activator on the coupling efficiency and overall coupling purity a single coupling reaction of L-DNA T was performed on a solid support onto which a 9-mer fully phosphorothioated DNA T previously had been synthesized with the use of stereorandom 2-cyanoethyl phosphoramidites. The synthesis took place on an AKTA OligoPilot 100 in 0.2 μmol scale. 2 coupling reactions (10 minutes coupling time) were performed with a thiolation and washing step in between the two coupling reactions, followed by thiolation, capping and detritylation. Hereafter, the solid support was treated with 20% Diethylamine in acetonitrile.

(133) The activator was selected from one of the following as indicated in the figure. A) 1 M 4,5-Dicyanoimidazole (DCI)+0.1 N-methylimidazole (NMI) B) 0.5 M Pyridinium hydrochloride in MeCN C) 0.2 M N-Phenyl imidazolium triflate in MeCN D) 0.2 M benzimidazolium triflate in MeCN+0.1 M NMI E) 0.2 M benzimidazolium triflate in MeCN L-DNA T was dissolved in a solution of 3.5% pyridine in MeCN at 0.15 M concentration. Sulfurization was carried out using 0.1 M Xanthane hydride in pyridine:MeCN (1:1), capping was carried out using 20% NMI in MeCN in Cap A and Pyridine/Ac2O/MeCN (3:5:2) as Cap B. Detritylation was carried out using 3% V/V dichloroacetic acid in dichloromethane

(134) The oligonucleotide was globally deprotected using concentrated aq. ammonium hydroxide at 55° C. for 24 h. The crude material was analyzed by UPLC-MS analysis. The chromatogram detected by UV absorbance at 260 nm primarily consisted of three peaks, the Full-length T10 oligomer, the T9 oligomer resulting from a failed coupling reaction and a T18 oligomer, resulting as impurity from the coupling reaction. These 3 peaks were quantified by integrating the absorbance of the peak at 260 nm, and are given below as a function of the activator used for the coupling reaction.

(135) TABLE-US-00001 % UV T9 % UV T10 % UV T18 Activator oligomer oligomer oligomer A 3.5% 86.5% 7.7% B 1.0% 94.7% 3.4% C 5.8% 86.8% 6.9% D 28.1% 67.3% 4.0% E 3.0% 92.2% 4.2%

(136) It is seen that the highest % UV of the desired T10 oligomer is obtained for activator B (0.5 M Pyridinium hydrochloride in MeCN) with the lowest level of T9 coupling failure species, and T18 impurity.

Example 2

(137) To examine the effect of the activator on the coupling efficiency and overall coupling purity a single coupling reaction of L-DNA T was performed on a solid support onto which a 15-mer fully phosphorothioated DNA T previously had been synthesized with the use of stereorandom 2-cyanoethyl phosphoramidites. The synthesis took place on a AKTA OligoPilot 100 in 0.2 μmol scale. 2 coupling reactions (10 minutes coupling time) were performed with a thiolation and washing step in between the two coupling reactions, followed by thiolation, capping and detritylation. Hereafter, the solid support was treated with 20% Diethylamine in acetonitrile.

(138) The activator was selected from one of the following as indicated in the figure. A) 1 M 4,5-Dicyanoimidazole (DCI)+0.1 N-methylimidazole (NMI) B) 0.5 M Pyridinium hydrochloride in MeCN C) 0.2 M N-Phenyl imidazolium triflate in MeCN D) 0.2 M benzimidazolium triflate in MeCN+0.1 M NMI E) 0.2 M benzimidazolium triflate in MeCN L-DNA T was dissolved in a solution of 3.5% pyridine in MeCN at 0.15 M concentration. Sulfurization was carried out using 0.1 M Xanthane hydride in pyridine:MeCN (1:1), capping was carried out using 20% NMI in MeCN in Cap A and Pyridine/Ac2O/MeCN (3:5:2) as Cap B. Detritylation was carried out using 3% V/V dichloroacetic acid in dichloromethane

(139) The oligonucleotide was globally deprotected using concentrated aq. ammonium hydroxide at 55° C. for 24 h. The crude material was analyzed by UPLC-MS analysis. The chromatogram detected by UV absorbance at 260 nm primarily consisted of three peaks, the Full-length T16 oligomer, the T15 oligomer resulting from a failed coupling reaction and a T30 oligomer, resulting as impurity from the coupling reaction. These 3 peaks were quantified by integrating the absorbance of the peak at 260 nm, and are given below as a function of the activator used for the coupling reaction.

(140) TABLE-US-00002 % UV T15 % UV T16 % UV T30 Activator oligomer oligomer oligomer A 5.2% 85.4% 9.4% B 1.8% 92.6% 4.2% E 6.0% 87.15 6.32

(141) It is seen that the highest % UV of the desired T16 oligomer is obtained for activator B (0.5 M Pyridinium hydrochloride in MeCN) with the lowest level of T15 coupling failure species, and T30 impurity.

Example 3

(142) To examine the effect of the activator on the coupling efficiency a single coupling reaction of L-LNA T was performed on a solid support onto which a 15-mer fully phosphorothioated DNA T previously had been synthesized with the use of stereorandom 2-cyanoethyl phosphoramidites. The synthesis took place on a AKTA OligoPilot 100 in 0.2 μmol scale. 2 coupling reactions (10 minutes coupling time) were performed with a thiolation and washing step in between the two coupling reactions, followed by thiolation, capping and detritylation. Hereafter, the solid support was treated with 20% Diethylamine in acetonitrile.

(143) The activator was selected from one of the following as indicated in the figure. A) 1 M 4,5-Dicyanoimidazole (DCI)+0.1 N-methylimidazole (NMI) B) 0.5 M Pyridinium hydrochloride in MeCN C) 0.25 M Pyridinium hydrochloride in MeCN D) 0.25 M Pyridinium hydrobromide in MeCN L-LNA T was dissolved in a solution of 3.5% pyridine in MeCN at 0.15 M concentration. Sulfurization was carried out using 0.1 M Xanthane hydride in pyridine:MeCN (1:1), capping was carried out using 20% NMI in MeCN in Cap A and Pyridine/Ac2O/MeCN (3:5:2) as Cap B. Detritylation was carried out using 3% V/V dichloroacetic acid in dichloromethane

(144) The oligonucleotide was globally deprotected using concentrated aq. ammonium hydroxide at 55° C. for 24 h. The crude material was analyzed by UPLC-MS analysis. The UV absorbance of the full length product peak was compared to the UV absorbance of the T15 peak, to obtain a relative measure of coupling efficiency.

(145) TABLE-US-00003 Activator Coupling efficiency A 71.2% B 91.9% C 77.3% D 96.9%

(146) It is seen that the highest coupling efficiency is achieved with activator D (0.25 M Pyridinium hydrobromide in MeCN), despite the concentration of the activator has been lowered to 0.25M compared to pyridinium hydrochloride.

Example 4

(147) To examine the effect of the activator on the coupling efficiency a single coupling reaction of L-LNA .sup.mC was performed on a solid support onto which a 15-mer fully phosphorothioated DNA T previously had been synthesized with the use of stereorandom 2-cyanoethyl phosphoramidites. The synthesis took place on a AKTA OligoPilot 100 in 0.2 μmol scale. 2 coupling reactions (10 minutes coupling time) were performed with a thiolation and washing step in between the two coupling reactions, followed by thiolation, capping and detritylation. Hereafter, the solid support was treated with 20% Diethylamine in acetonitrile.

(148) The activator was selected from one of the following as indicated in the figure. A) 1 M 4,5-Dicyanoimidazole (DCI)+0.1 N-methylimidazole (NMI) B) 0.5 M Pyridinium hydrochloride in MeCN C) 0.25 M Pyridinium hydrobromide in MeCN L-LNA .sup.mC was dissolved in a solution of 3.5% pyridine in MeCN at 0.15 M concentration. Sulfurization was carried out using 0.1 M Xanthane hydride in pyridine:MeCN (1:1), capping was carried out using 20% NMI in MeCN in Cap A and Pyridine/Ac2O/MeCN (3:5:2) as Cap B. Detritylation was carried out using 3% V/V dichloroacetic acid in dichloromethane

(149) The oligonucleotide was globally deprotected using concentrated aq. ammonium hydroxide at 55° C. for 24 h. The crude material was analyzed by UPLC-MS analysis. The UV absorbance of the full length product peak was compared to the UV absorbance of the T15 peak, to obtain a relative measure of coupling efficiency.

(150) TABLE-US-00004 Activator Coupling efficiency A 53.3% B 76.8% C 83.6%

(151) It is seen that the highest coupling efficiency is achieved with activator C (0.25 M Pyridinium hydrobromide in MeCN).

Example 5

(152) To examine the effect of the activator on the coupling efficiency a single coupling reaction of L-LNA .sup.mC was performed on a solid support onto which a 15-mer fully phosphorothioated DNA T previously had been synthesized with the use of stereorandom 2-cyanoethyl phosphoramidites. The synthesis took place on a AKTA OligoPilot 100 in 0.2 μmol scale. 2 coupling reactions (20 minutes coupling time) were performed with a thiolation and washing step in between the two coupling reactions, followed by thiolation, capping and detritylation. Hereafter, the solid support was treated with 20% Diethylamine in acetonitrile.

(153) The activator was selected from one of the following as indicated in the figure. A) 0.5 M Pyridinium hydrochloride in MeCN B) 0.25 M Pyridinium hydrobromide in MeCN L-LNA .sup.mC was dissolved in a solution of 3.5% pyridine in MeCN at 0.15 M concentration. Sulfurization was carried out using 0.1 M Xanthane hydride in pyridine:MeCN (1:1), capping was carried out using 20% NMI in MeCN in Cap A and Pyridine/Ac2O/MeCN (3:5:2) as Cap B. Detritylation was carried out using 3% V/V dichloroacetic acid in dichloromethane

(154) The oligonucleotide was globally deprotected using concentrated aq. ammonium hydroxide at 55° C. for 24 h. The crude material was analyzed by UPLC-MS analysis. The UV absorbance of the full length product peak was compared to the UV absorbance of the T15 peak, to obtain a relative measure of coupling efficiency.

(155) TABLE-US-00005 Activator Coupling efficiency A 87.1% B 91.0%

(156) It is seen that the highest coupling efficiency is achieved with activator B (0.25 M Pyridinium hydrobromide in MeCN), and it is also seen that an increased coupling time leads to increased coupling efficiencies compared to example 4.

Example 6

(157) To examine the effect of the activator on the coupling efficiency a single coupling reaction of L-LNA .sup.mC was performed on a solid support onto which a 15-mer fully phosphorthioated DNA T previously had been synthesized with the use of stereorandom 2-cyanoethyl phosphoramidites. The synthesis took place on a AKTA OligoPilot 100 in 0.2 μmol scale. 2 coupling reactions (20 minutes coupling time) were performed with a thiolation and washing step in between the two coupling reactions, followed by thiolation, capping and detritylation. Hereafter, the solid support was treated with 20% Diethylamine in acetonitrile.

(158) The activator was selected from one of the following as indicated in the figure. A) 0.5 M Pyridinium hydrochloride in MeCN B) 0.25 M Pyridinium hydrobromide in MeCN C) 0.25M Pyridinium trifluoroacetate D) 0.25M Pyridinium para-toluenesulfonate E) 0.25 M Pyridinium triflate L-LNA .sup.mC was dissolved in a solution of 3.5% pyridine in MeCN at 0.15 M concentration. Sulfurization was carried out using 0.1 M Xanthane hydride in pyridine:MeCN (1:1), capping was carried out using 20% NMI in MeCN in Cap A and Pyridine/Ac2O/MeCN (3:5:2) as Cap B. Detritylation was carried out using 3% V/V dichloroacetic acid in dichloromethane

(159) The oligonucleotide was globally deprotected using concentrated aq. ammonium hydroxide at 55° C. for 24 h. The crude material was analyzed by UPLC-MS analysis. The UV absorbance of the full length product peak was compared to the UV absorbance of the T15 peak, to obtain a relative measure of coupling efficiency.

(160) TABLE-US-00006 Activator Coupling efficiency A 87.1% B 91.0% C 68.7% D 81.8% E 80.3%

Example 7

(161) A 50 μmol scale synthesis was carried out using 393 μmol/g Kinnovate resine on an Äkta OligoPilot 10. Using conventional 5′-dimethoxytrityl-3′-β-cyanoethyl phosphoramidites, 12 stereorandom couplings 5′-CGATCGATCGAT-3′ were carried out, followed by four stereodefined couplings (C*A*G*T*) depicted bellow. All stereodefined monomers were dissolved in 3.5% pyridine in acetonitrile

(162) ##STR00047##

(163) All synthetic steps were carried out according to a standard oligonucleotides synthesis: Detritylation Coupling Oxydation/Sulphurization Capping
Reagents used in the synthetic cycle: Deblock: 3% Dichloroacetic acid in toluene (v/v) Activator: see table below Stereorandom phosphoramidites: 0.2 M solution in Acetonitrile Stereodefined phosphoramidites: see table below Cap A: N-Methylimidazole/Acetonitrile 2/8 (v/v) Cap B1: Acetic Anhydride/Acetonitrile 4/6 (v/v) Cap B2: Pyridine/Acetonitrile 6/4 (v/v) Sulfurizing reagent: 0.1M Xanthane hydride in Acetonitrile/Pyridine 1/1 (v/v) Oxidizer: Iodine/Water/Pyridine 12.7/1/9 (w/v/v)

(164) After completion of the oligonucleotides synthesis, a deprotection/cleavage step is carried out in a solution of 32% Ammonia in water at 65° C. for 6 h. The results of the optimization are depicted in the table below:

(165) In the table below the sequence of coupling, oxidation, wash (COW) was used in cases where more than one coupling was performed.

(166) TABLE-US-00007 Recy. Yield Activators Equivalents (a/c/g/t resp.) (m) (%) PyrBr 0.25M  2 × 2eq. 2 × 2eq. 2 × 2eq. 2 × 2eq. 10 15 PyrOtf 0.25M  2 × 2eq. 2 × 2eq. 2 × 2eq. 2 × 2eq. 10 8 PyrOTf   1M 1 × 5eq. 1 × 5eq. 1 × 5eq. 1 × 5eq. 10 23 PyrBr 0.5M 1 × 5eq. 1 × 5eq. 1 × 5eq. 1 × 5eq. 10 11 CMPT   1M 1 × 5eq. 1 × 5eq. 1 × 5eq. 1 × 5eq. 10 18 PyrOTf 0.5M 1 × 5eq. 1 × 5eq. 1 × 5eq. 1 × 5eq. 10 17 PyrOTf 0.3M 1 × 5eq. 1 × 5eq. 1 × 5eq. 1 × 5eq. 10 23 PyrBr 0.3M 1 × 5eq. 1 × 5eq. 1 × 5eq. 1 × 5eq. 10 22 PyrOTf 0.3M 1 × 5eq. 1 × 5eq. 1 × 5eq. 1 × 5eq. 20 24

(167) CMPT: N-(Cyanomethyl)-pyrrolidinium triflate

(168) Pyr: Pyridinium

(169) OTf: Trifluoromethane sulfonate