Method for the preparation of oligonucleotides using a modified oxidation protocol

Abstract

The present invention relates to a method for the preparation of mixed P=O / P=S backbone oligonucleotides, which comprises selectively oxidizing an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II according to a scheme applying a novel oxidation protocol, and to a new oxidation solution. TIFF2023533017000011.tif47140

Description

[Technical Field]

[0001] The present invention provides a novel method for the preparation of mixed P=O / P=S backbone oligonucleotides, comprising the scheme The present invention relates to a novel method for the preparation of mixed P=O / P=S backbone oligonucleotides, comprising oxidizing an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II according to TIFF0007777576000001.tif47140, wherein the oxidation utilizes specific and novel oxidation solutions. [Background technology]

[0002] Oligonucleotide synthesis is essentially the stepwise addition of nucleotide residues to the 5' end of a growing chain until the desired sequence is assembled.

[0003] Generally, each addition is called a synthesis cycle and consists of the following chemical reactions: a 1) deblocking protected hydroxyl groups on the solid support; a2) coupling a first nucleoside as an activated phosphoramidite to a free hydroxyl group on a solid support; a3) oxidizing or sulfurizing each P-linked nucleoside (phosphite triester) to form the respective phosphodiester (P=O) or the respective phosphorothioate (P=S); a4) optionally capping any unreacted hydroxyl groups on the solid support; a5) deblocking the 5' hydroxyl group of the first nucleoside bound to the solid support; a6) coupling a second nucleoside as an activated phosphoramidite to form the respective P-linked dimer; a7) oxidizing or sulfurizing each of the P-linked dinucleotides (phosphite triesters) to form the respective phosphodiesters (P=O) or the respective phosphorothioates (P=S); a8) optionally capping any unreacted 5' hydroxyl groups; a9) repeating the above steps a5 to a8 until the desired sequence is assembled.

[0004] The oxidation step is typically carried out using an oxidation solution containing iodine, an organic solvent, typically pyridine, and water.

[0005] However, it has been observed that application of the freshly prepared oxidizing solution not only results in the desired oxidation of the intermediate phosphite triester compound of formula I to the phosphodiester compound of formula II, but as a side reaction, the phosphorothioate internucleotide linkages present in the molecule are affected by the conversion of P=S to P=O at the internucleotide bond, which can result in a higher than expected content of phosphodiester linkages in the compound of formula II. Summary of the Invention

[0006] It was therefore an object of the present invention to find an oxidation protocol that allows the selective oxidation of phosphite triester compounds of formula I to phosphodiester compounds of formula II without affecting the phosphorothioate internucleotide linkages. A further object of the present invention was to find an oxidation solution that can be easily applied when prepared without the need for further treatment, such as aging.

[0007] The object of the present invention is a method for the preparation of mixed P=O / P=S backbone oligonucleotides, which comprises the steps of: It has been found that this can be achieved by a process for the preparation of mixed P=O / P=S backbone oligonucleotides, which comprises oxidizing an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II according to TIFF0007777576000002.tif47140, characterized in that the oxidation solution further contains iodide. [The present invention 1001] A method for the preparation of mixed P=O / P=S backbone oligonucleotides, comprising the steps of: TIFF0007777576000003.tif47140 oxidizing an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II according to The oxidizing solution further contains iodide. The method. [The present invention 1002] 1001. The process of claim 1001, wherein said iodide is selected from hydrogen iodide, alkali iodide, or alkali triiodide. [The present invention 1003] 1001 or 1002, wherein the iodide is selected from hydrogen iodide or alkali iodide. [The present invention 1004] The method according to any one of claims 1001 to 1003, wherein the iodide is selected from alkali iodides. [The present invention 1005] The process according to any one of claims 1001 to 1004, wherein the molar ratio of iodine to iodide in said oxidizing solution is selected in the range of 1.0:0.1 to 1.0:3.0, preferably 1.0:1.0 to 1.0:2.0. [The present invention 1006] The organic solvent may be pyridine or C 1~6 The method according to any one of claims 1001 to 1005, wherein the alkyl group is selected from alkyl-substituted pyridines, preferably pyridine. [The present invention 1007] 1006. The process of claim 1006, wherein said organic solvent is selected from pyridine. [The present invention 1008] The process according to any one of claims 1001 to 1007, wherein the volume ratio of the organic solvent to water is 1:1 to 20:1, preferably 5:1 to 15:1, more preferably 9:1. [The present invention 1009] The method according to any one of claims 1001 to 1008, wherein the iodine concentration in the oxidizing solution is 10 mM to 100 mM, preferably 15 mM to 60 mM. [The present invention 1010] 1009. The method of any one of claims 1001 to 1009, wherein an oxidizing solution is used, said oxidizing solution having an iodine content of 50 mM, and iodide is added to said oxidizing solution until said oxidizing solution has a conductivity of 1500 μS / cm or more. [The present invention 1011] 50mM KI and 50mM I 2 The method according to any one of claims 1001 to 1010, wherein an oxidizing solution having a conductivity of 1500 μS / cm or more, preferably 1650 to 2050 μS / cm, based on the content of [The present invention 1012] 1009. The method of any one of claims 1001 to 1009, wherein an oxidizing solution is used, said oxidizing solution having an iodine content of 10 mM, and iodide is added to said oxidizing solution until said oxidizing solution has a conductivity of 300 μS / cm or more. [The present invention 1013] 10 mM KI and 10 mM I 2 The method of any one of inventions 1001 to 1009 and 1012, wherein an oxidizing solution having a conductivity of 300 μS / cm or more, preferably 350 to 550 μS / cm, based on the content of [The present invention 1014] 1009. The method of any one of claims 1001 to 1009, wherein an oxidizing solution is used, said oxidizing solution having an iodine content of 20 mM, and iodide is added to said oxidizing solution until said oxidizing solution has a conductivity of 600 μS / cm or more. [The present invention 1015] 20 mM KI and 20 mM I 2 The method of any one of inventions 1001 to 1009 and 1014, wherein an oxidizing solution having a conductivity of 600 μS / cm or more, preferably 750 to 950 μS / cm, based on the content of [The present invention 1016] 1009. The method of any one of claims 1001 to 1009, wherein an oxidizing solution is used, said oxidizing solution having an iodine content of 100 mM, and iodide is added to said oxidizing solution until said oxidizing solution has a conductivity of 3000 μS / cm or more. [The present invention 1017] 100mM KI and 100mM I 2 The method of any one of inventions 1001 to 1009 and 1016, wherein an oxidizing solution having a conductivity of 3000 μS / cm or more, preferably 3200 to 3900 μS / cm, based on the content of [The present invention 1018] 10. The process of any one of claims 1001 to 1017, wherein the oxidation solution is capable of oxidizing the intermediate phosphite triester compound of formula I to the phosphodiester compound of formula II such that the P=O content in the reaction solution reaches a value of less than 2.5%, preferably less than 2.0%. [The present invention 1019] The method according to any one of claims 1001 to 1018, wherein the amount of iodine used in preparing the oxidizing solution is selected from the range of 1.1 equivalents to 15 equivalents, more preferably 1.5 equivalents to 4.5 equivalents. [The present invention 1020] The process of any one of claims 1001 to 1019, wherein the reaction temperature for the oxidation reaction is selected between 15°C and 27°C, more preferably between 18°C ​​and 24°C. [The present invention 1021] The method of any of claims 1001 to 1020, wherein said oligonucleotide consists of optionally modified DNA or RNA nucleoside monomers, or a combination thereof, and is 10 to 40, preferably 10 to 25 nucleotides in length. [The present invention 1022] a) 10-100 mM iodine; b) 0.1 to 3.0 molar equivalents of iodide relative to 1.0 molar equivalent of iodine; c) an organic solvent; d) Water and Including, The volume ratio of organic solvent to water is 20:1 to 1:1; Oxidizing solution. [The present invention 1023] a) 15-60 mM iodine; b) 1.0 to 2.0 molar equivalents of iodide per 1.0 molar equivalent of iodine; c) an organic solvent; d) Water and Including, The volume ratio of organic solvent to water is 5:1 to 15:1; The oxidizing solution of the present invention 1022. [The present invention 1024] a) 15-60 mM iodine; b) 1.0 to 2.0 molar equivalents of hydrogen iodide or alkali iodide relative to 1.0 molar equivalent of iodine; c) pyridine, d) Water and Including, The volume ratio of organic solvent to water is 5:1 to 15:1; The oxidizing solution of the present invention 1022 or 1023. [The present invention 1025] 1. A method for assessing the quality of an oxidizing solution, comprising: a) providing an oxidizing solution comprising iodine, an organic solvent, and water; b) measuring the conductivity of the oxidizing solution; c) evaluating the suitability of the oxidation solution for oxidizing an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II based on a particular threshold value of the measured conductivity; The method comprising: [The present invention 1026] 1026. The method of claim 1025, wherein the oxidizing solution further comprises iodide. DETAILED DESCRIPTION OF THE INVENTION

[0008] The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.

[0009] "C 1~6 The term "-alkyl" denotes a monovalent linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms, and more particularly 1 to 4 carbon atoms. Typical examples include methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, sec-butyl or t-butyl, preferably methyl or ethyl.

[0010] As used herein, the term "oligonucleotide" is defined as commonly understood by those skilled in the art as a molecule comprising two or more covalently linked nucleotides. For use as therapeutically valuable oligonucleotides, oligonucleotides are typically synthesized as lengths of 10 to 40 nucleotides, preferably 10 to 25 nucleotides.

[0011] Oligonucleotides can be composed of optionally modified DNA or RNA nucleoside monomers or combinations thereof.

[0012] As used herein, optionally modified refers to a nucleoside that is modified compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications in the sugar or nucleobase moieties.

[0013] Exemplary modifications may be 2'-O-(2-methoxyethyl)-substitution (2'-MOE) of the sugar moiety or locked nucleic acid (LNA), which is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen to the 4' carbon.

[0014] The term modified nucleoside may also be used interchangeably with the terms "nucleoside analogue" or modified "unit" or modified "monomer."

[0015] DNA or RNA nucleotides are generally linked by phosphodiester (P=O) or phosphorothioate (P=S) internucleotide bonds, which covalently link two nucleotides to one another.

[0016] According to the present invention, at least one internucleotide linkage must consist of phosphorothioate (P=S). Thus, in some oligonucleotides, all other internucleotide linkages may consist of phosphodiester (P=O), or in other oligonucleotides, the sequence of internucleotide linkages may vary and include both phosphodiester (P=O) and phosphorothioate (P=S) internucleotide linkages.

[0017] Thus, the term mixed P=O / P=S backbone oligonucleotide refers to an oligonucleotide in which at least one internucleotide linkage must consist of phosphorothioate (P=S) and at least one internucleotide linkage consists of phosphodiester (P=O).

[0018] Nucleobase moieties may be designated by the letter code for each corresponding nucleobase, e.g., A, T, G, C, or U, where each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are designated by capital letters A, T, G, and U for LNA nucleosides. Me C (5-methylcytosine) and lowercase a, t, g, c, and MeC. Modified nucleobases include, but are not limited to, nucleobases bearing protecting groups such as tert-butylphenoxyacetyl, phenoxyacetyl, benzoyl, acetyl, isobutyryl, or dimethylformamidino (see Wikipedia, Phosphoramidit-Synthese, https: / / de.wikipedia.org / wiki / Phosphoramidit-Synthese, March 24, 2016).

[0019] Preferably, the oligonucleotide consists of optionally modified DNA or RNA nucleoside monomers or combinations thereof and is 10 to 40, preferably 10 to 25 nucleotides in length.

[0020] The principles of oligonucleotide synthesis are well known in the art (see, e.g., Oligonucleotide synthesis in Wikipedia, the free encyclopedia, https: / / en.wikipedia.org / wiki / Oligonucleotide synthesis, March 15, 2016).

[0021] Today, large scale oligonucleotide synthesis is performed in an automated manner using computer-controlled synthesizers.

[0022] In principle, oligonucleotide synthesis is a solid-phase synthesis, in which the oligonucleotides to be assembled are covalently attached to a solid support material via their 3'-terminal hydroxy groups and remain attached there throughout the chain assembly. Suitable supports are commercially available macroporous polystyrene supports such as GE Healthcare's Primer support 5G or Kinovate's NittoPhase® HL support.

[0023] Subsequent cleavage from the resin can be achieved using concentrated aqueous ammonia. Protecting groups on the phosphate and nucleotide bases are also removed during this cleavage procedure.

[0024] As outlined above, the method for the preparation of mixed P=O / P=S backbone oligonucleotides involves the oxidation of an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II.

[0025] The oxidizing solution can be prepared by mixing iodide with water and an organic solvent and then adding iodine.

[0026] The iodide may be selected from hydrogen iodide, alkali iodides or alkali triiodides, preferably from hydrogen iodide or alkali iodide, more preferably from sodium iodide or potassium iodide.

[0027] The organic solvent is pyridine or C 1~6 It may be selected from alkyl substituted pyridines such as lutidine, but preferably pyridine.A further organic solvent such as tetrahydrofuran may be present.

[0028] The volume ratio of the organic solvent to water is selected in principle from 1:1 to 20:1, preferably from 5:1 to 15:1, and more preferably is 9:1.

[0029] The molar ratio of iodine to iodide in the oxidation solution is selected in the range of 1.0:0.1 to 1.0:3.0, preferably 1.0:1.0 to 1.0:2.0.

[0030] The iodine concentration in the oxidation solution is typically applied in the range of 10 mM to 100 mM, preferably 15 mM to 60 mM.

[0031] Iodide is added until the oxidizing solution has a conductivity of ≥ 1500 μS / cm, based on an iodine content of 50 mM.

[0032] In a preferred embodiment, the iodide is potassium iodide and the oxidizing solution has a conductivity of 1500 μS / cm or more, preferably 1650-2050 μS / cm, more preferably 1750-1950 μS / cm, based on a content of 50 mM KI and 50 mM I2.

[0033] Iodide is added until the oxidizing solution has a conductivity of ≥ 300 μS / cm, based on an iodine content of 10 mM.

[0034] In a preferred embodiment, the iodide is potassium iodide and the oxidizing solution has a conductivity of 300 μS / cm or greater, preferably 350-550 μS / cm, more preferably 400-500 μS / cm, based on 10 mM KI and 10 mM I2.

[0035] Iodide is added until the oxidizing solution has a conductivity of ≥ 600 μS / cm, based on an iodine content of 20 mM.

[0036] In a preferred embodiment, the iodide is potassium iodide and the oxidizing solution has a conductivity of 600 μS / cm or greater, preferably 750-950 μS / cm, more preferably 800-900 μS / cm, based on 20 mM KI and 20 mM I2.

[0037] Iodide is added until the oxidizing solution has a conductivity of ≥ 3000 μS / cm, based on an iodine content of 100 mM.

[0038] In a preferred embodiment, the iodide is potassium iodide and the oxidizing solution has a conductivity of 3000 μS / cm or greater, preferably 3200-3900 μS / cm, more preferably 3350-3750 μS / cm, based on 100 mM KI and 100 mM I2.

[0039] Typically, the oxidation solution is capable of oxidizing the intermediate phosphite triester compound of formula I to the phosphodiester compound of formula II such that the P=O content in the reaction solution reaches a value of less than 2.5%, preferably less than 2.0%.

[0040] A further embodiment of the present invention is a method for assessing the quality of an oxidizing solution, comprising the steps of: a) providing an oxidizing solution comprising iodine, an organic solvent, and water; b) measuring the conductivity of the oxidizing solution; c) evaluating the suitability of the oxidation solution for oxidizing the intermediate phosphite triester compound of formula I to the phosphodiester compound of formula II based on a specific threshold value of the measured conductivity; A method is provided, comprising:

[0041] In a further more preferred embodiment of the method for assessing the quality of an oxidizing solution, the oxidizing solution further comprises iodide.

[0042] The amount of iodine used in the preparation for the oxidation reaction is usually selected from the range of 1.1 to 15 equivalents, more preferably 1.5 to 4.5 equivalents.

[0043] The oxidation reaction is carried out at a temperature between 15°C and 27°C, more preferably between 18°C ​​and 24°C.

[0044] As outlined above, in preferred embodiments of the present invention, i.e., in a stoichiometric ratio of iodine to iodide or in a ratio where an excess of iodide is present, the oxidizing solution can be applied immediately after its preparation.

[0045] In another, but less preferred, embodiment of the present invention, a ratio of iodine to iodide can be used with less than a stoichiometric amount of iodide.

[0046] Such oxidizing solutions may require aging for a period of time before they have the required properties in terms of conductivity and ability to selectively oxidize phosphite triester compounds of formula I to phosphodiester compounds of formula II.

[0047] The optimum period of aging is largely determined by the temperature at which the oxidizing solution is aged: lower aging temperatures result in longer aging periods, while higher aging temperatures significantly shorten the aging time.

[0048] For example, the oxidizing solution can be aged at a temperature of 20°C to 100°C, preferably at a temperature of 30°C to 60°C.

[0049] The time required for aging the oxidizing solution must be sufficient to result in the selective oxidation of the phosphite triester compound of formula I to the phosphodiester compound of formula II without affecting the phosphorothioate internucleotide linkages.

[0050] Generally, the oxidizing solution can be aged for at least 1 day, 3 days, 5 days, 10 days, 15 days, or at least 20 days.

[0051] The period can vary greatly depending on the aging temperature, as mentioned above; at aging temperatures of 30°C to 35°C, it can vary from 10 to 150 days, more typically 20 to 60 days, while at aging temperatures of 60°C to 65°C, it can vary from 1 to 30 days, more typically 2 to 15 days.

[0052] Aging is generally accompanied by an increase in conductivity (μS / cm) and a decrease in pH until a certain plateau is reached.

[0053] In a further embodiment, the present invention provides a method for treating a cancer cell comprising: a) 10-100 mM iodine; b) 0.1 to 3.0 molar equivalents of iodide relative to 1.0 molar equivalent of iodine; c) an organic solvent; d) Water and wherein the volume ratio of organic solvent to water is 20:1 to 1:1; Preferably, a) 15-60 mM iodine; b) 1.0 to 2.0 molar equivalents of iodide per 1.0 molar equivalent of iodine; c) an organic solvent; d) Water and wherein the volume ratio of organic solvent to water is 5:1 to 15:1; More preferably, a) 15-60 mM iodine; b) 1.0 to 2.0 molar equivalents of hydrogen iodide or alkali iodide relative to 1.0 molar equivalent of iodine; c) pyridine, d) Water and wherein the volume ratio of pyridine to water is 5:1 to 15:1; Even more preferably, a) 15-60 mM iodine; b) 1.0 to 2.0 molar equivalents of sodium iodide or potassium iodide per 1.0 molar equivalent of iodine; c) pyridine, d) Water and wherein the volume ratio of pyridine to water is 9:1.

[0054] Illustratively, the oligonucleotide may be: Includes a new oxidizing solution that can be selected from TIFF0007777576000004.tif5140.

[0055] The underlined residues are 2'-MOE nucleosides. The positions of the phosphorothioate and phosphate diester bonds are indicated by S and O, respectively. Note that 2'-O-(2-methoxyethyl)-5-methyluridine (2'-MOE MeU) nucleosides are sometimes referred to as 2'-O-(2-methoxyethyl)ribothymidine (2'-MOE T).

[0056] The compounds disclosed herein have the following nucleobase sequences: SEQ ID NO: 1: cucagtaacattgacaccac [Example]

[0057] Composite of TIFF0007777576000005.tif5146

[0058] Oligonucleotides were prepared on the solid phase using standard phosphoramidite chemistry on a 2.20 mmol scale using an AKTA Oligopilot 100 and a Primer Support Unylinker (NittoPhase LH Unylinker 330). Typically, 1.4 equivalents of DNA / MOE-phosphoramidite were used. Other reagents received from commercial sources (dichloroacetic acid, 1-methylimidazole, 4,5-dicyanoimidazole, acetic anhydride, phenylacetyl disulfide, pyridine, triethylamine) were used to prepare reagent solutions of appropriate concentrations (see details below). An oxidant solution was freshly prepared (see below). Cleavage and deprotection were performed using ammonium hydroxide to obtain crude oligonucleotides.

[0059] Standard Reagent Solution TIFF0007777576000006.tif59170

[0060] Preparation of iodine / potassium iodide solution Potassium iodide was added to water at room temperature, followed by pyridine. Iodine was added, and the mixture was stirred under a positive pressure of dry nitrogen for 1 hour before use. TIFF0007777576000007.tif42170

[0061] Preparation of iodine / sodium iodide solution 7.49 g of sodium iodide was added to 101 g of water at room temperature, followed by 886 g of pyridine. 12.7 g of iodine was added, and the mixture was stirred under a positive pressure of dry nitrogen for 1 hour before use.

[0062] Oxidation examples using different oxidizer solutions without aging TIFF0007777576000008.tif107168 1refers to the percentage of molecules with a mass difference of 16 Da relative to the molecular mass of the desired compound as determined by mass spectrometry, i.e., the percentage of molecules in which one P=S bond has been converted to a P=O bond.

[0063] Aging of KI (50 mM) / I2 (50 mM) solution at 30-35°C The solution was stored in an amber glass bottle at 30-35°C until use.

[0064] Oxidation example using aged (30-35°C) KI (50 mM) / I2 (50 mM) solution TIFF0007777576000009.tif53168 1 refers to the percentage of molecules with a mass difference of 16 Da relative to the molecular mass of the desired compound as determined by mass spectrometry, i.e., the percentage of molecules in which one P=S bond has been converted to a P=O bond.

Claims

1. A method for the preparation of mixed P=O / P=S backbone oligonucleotides, comprising the steps of: starting with an oligonucleotide in which at least one internucleotide bond is phosphorothioate (P=S), the method comprises oxidizing an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II, The oxidizing solution further contains iodide. The method.

2. 2. The method of claim 1, wherein the iodide is selected from hydrogen iodide, alkali iodide, or alkali triiodide.

3. 3. The method according to claim 1, wherein the iodide is selected from hydrogen iodide or from alkali iodides.

4. 4. The method according to any one of claims 1 to 3, wherein the iodide is selected from alkali iodides.

5. 5. The method according to any one of claims 1 to 4, wherein the molar ratio of iodine to iodide in the oxidizing solution is selected in the range of 1.0:0.1 to 1.0:3.

0.

6. The method of claim 1, wherein the molar ratio of iodine to iodide in the oxidizing solution is selected in the range of 1.0:1.0 to 1.0:2.

0.

7. The organic solvent may be pyridine or C １～６ The method of any one of claims 1 to 6, wherein the alkyl group is selected from alkyl-substituted pyridines.

8. 8. The method of claim 7, wherein the organic solvent is selected from pyridine.

9. 9. The method according to any one of claims 1 to 8, wherein the volume ratio of organic solvent to water is from 1:1 to 20:

1.

10. The method of claim 1, wherein the volume ratio of organic solvent to water is 5:1 to 15:

1.

11. The method of claim 1, wherein the volume ratio of organic solvent to water is 9:

1.

12. 12. The method according to any one of claims 1 to 11, wherein the iodine concentration in the oxidizing solution is from 10 mM to 100 mM.

13. The method of claim 1, wherein the iodine concentration in the oxidizing solution is 15 mM to 60 mM.

14. 14. The method according to any one of claims 1 to 13, wherein an oxidizing solution is used, said oxidizing solution having an iodine content of 50 mM, and iodide is added to said oxidizing solution until said oxidizing solution has a conductivity of 1500 μS / cm or more.

15. 50 mM KI and 50 mM I ２ 15. The method according to claim 1, wherein an oxidizing solution is used having a conductivity of 1500 μS / cm or more based on the content of 16. The method according to any one of claims 1 to 14, wherein an oxidizing solution is used having a conductivity of 1650 to 2050 μS / cm based on a content of 50 mM KI and 50 mM I 2 .

17. 14. The method according to any one of claims 1 to 13, wherein an oxidizing solution is used, said oxidizing solution having an iodine content of 10 mM, and iodide is added to said oxidizing solution until said oxidizing solution has a conductivity of 300 μS / cm or more.

18. 10 mM KI and 10 mM I ２ 18. The method according to claim 1, wherein an oxidizing solution is used having a conductivity of 300 μS / cm or more based on the content of 19. The method according to any one of claims 1 to 13 and 17, wherein an oxidizing solution is used having a conductivity of 350 to 550 μS / cm based on a content of 10 mM KI and 10 mM I 2 .

20. 14. The method according to any one of claims 1 to 13, wherein an oxidizing solution is used, said oxidizing solution having an iodine content of 20 mM, and iodide is added to said oxidizing solution until said oxidizing solution has a conductivity of 600 μS / cm or more.

21. 20 mM KI and 20 mM I ２ 21. The method according to claim 1, wherein an oxidizing solution is used having a conductivity of 600 μS / cm or more based on the content of 22. The method according to any one of claims 1 to 13 and 20, wherein an oxidizing solution is used having a conductivity of 750 to 950 μS / cm based on a content of 20 mM KI and 20 mM I 2 .

23. 14. The method according to any one of claims 1 to 13, wherein an oxidizing solution is used, said oxidizing solution having an iodine content of 100 mM, and iodide is added to said oxidizing solution until said oxidizing solution has a conductivity of 3000 μS / cm or more.

24. 100 mM KI and 100 mM I ２ 24. The method according to claim 1, wherein an oxidizing solution is used having a conductivity of 3000 μS / cm or more based on the content of 25. The method according to any one of claims 1 to 13 and 23, wherein an oxidizing solution is used having a conductivity of 3200 to 3900 μS / cm based on a content of 100 mM KI and 100 mM I 2 .

26. 26. The method of any one of claims 1 to 25, wherein the oxidation solution is capable of oxidizing the intermediate phosphite triester compound of formula I to the phosphodiester compound of formula II such that the P=O content in the reaction solution reaches a value of less than 2.5%.

27. ​​A method described in any one of claims 1 to 25, wherein the oxidation solution is capable of oxidizing the intermediate phosphite triester compound of formula I to the phosphodiester compound of formula II so that the P=O content in the reaction solution reaches a value of less than 2.0%.

28. 28. The method according to any one of claims 1 to 27, wherein the amount of iodine used in preparing the oxidizing solution is selected between 1.1 equivalents and 15 equivalents.

29. A method according to any one of claims 1 to 27, wherein the amount of iodine used in preparing the oxidizing solution is selected between 1.5 equivalents and 4.5 equivalents.

30. The method according to any one of claims 1 to 29, wherein the reaction temperature for the oxidation reaction is selected between 15°C and 27°C.

31. The method of claim 1, wherein the reaction temperature for the oxidation reaction is selected between 18°C ​​and 24°C.

32. 32. The method of any one of claims 1 to 31, wherein the oligonucleotide consists of optionally modified DNA or RNA nucleoside monomers, or a combination thereof, and is 10 to 40 nucleotides in length.

33. The method of any one of claims 1 to 31, wherein the oligonucleotide is composed of optionally modified DNA or RNA nucleoside monomers, or a combination thereof, and is 10 to 25 nucleotides in length.

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