Method for preparing oligonucleotides using an improved oxidation scheme

Abstract

The present invention relates to a method for producing mixed P=O / P=S backbone oligonucleotides, the method comprising applying a novel oxidation scheme and a novel oxidation solution to selectively oxidize an intermediate phosphite compound of formula I to a phosphodiester compound of formula II, according to the scheme.

Description

[0001] This invention relates to a novel method for producing 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 a scheme.

[0002]

[0003] The oxidation process utilizes specific oxidation solutions and novel oxidation solutions.

[0004] Oligonucleotide synthesis is, in principle, the stepwise addition of nucleotide residues to the 5'-end of the growth chain until the desired sequence is assembled.

[0005] Typically, each addition is called a synthesis cycle, and in principle, it consists of a chemical reaction.

[0006] a1) Unblock the protected hydroxyl groups on the solid support.

[0007] a2) The first nucleoside, which serves as the activator of phosphoramidite, is coupled to the free hydroxyl group on the solid support.

[0008] a3) Oxidize or sulfide the corresponding P-linked nucleoside (phosphite) to form the corresponding phosphate diester (P=O) or the corresponding thiophosphate (P=S);

[0009] a4) Optionally, end any unreacted hydroxyl groups on the solid support;

[0010] a5) Unblocks the 5' hydroxyl group of the first nucleoside attached to the solid support;

[0011] a6) The second nucleoside of the activated phosphoramidite will be coupled to form the corresponding P-linked dimer;

[0012] a7) Oxidize or sulfide the corresponding P-linked dinucleotide (phosphite) to form the corresponding phosphodiester (P=O) or the corresponding thiophosphate (P=S).

[0013] a8) Optionally, end-cap any unreacted 5' hydroxyl group;

[0014] a9) Repeat steps a5 to a8 until the desired sequence is assembled.

[0015] The oxidation step is typically carried out using an oxidation solution containing iodine, an organic solvent (usually pyridine), and water.

[0016] However, it was observed that when the freshly prepared oxidation solution was applied, not only did the desired intermediate phosphite compound of Formula I oxidize to the phosphodiester compound of Formula II, but also, as a side reaction, the internucleotide bonds of the thiophosphate ester present in the molecule may be affected by the P=S to P=O conversion at the internucleotide bond sites, which resulted in a higher than expected content of phosphodiester bonds in the compound of Formula II.

[0017] Therefore, an object of the present invention is to find an oxidation scheme that allows the selective oxidation of a phosphite triester compound of formula I to a phosphodiester compound of formula II without affecting the internucleotide bonding of thiophosphates. Another object of the present invention is to find an oxidation solution that can be readily applied without further treatment (such as aging) during preparation.

[0018] It has been discovered that the objectives of this invention can be achieved by producing mixed P=O / P=S main-chain oligonucleotides, a method comprising oxidizing an intermediate phosphite compound of formula I into a phosphodiester compound of formula II using an oxidizing solution containing iodine, an organic solvent, and water, according to a scheme.

[0019]

[0020] Furthermore, it is characterized by the presence of an additional iodide in the oxidizing solution.

[0021] The following definitions are set forth to illustrate and define the meaning and scope of various terms used to describe the present invention.

[0022] Term "C" 1-6 "-alkyl" refers to a monovalent straight-chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms, and in more specific embodiments 1 to 4 carbon atoms. Typical examples include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl, preferably methyl or ethyl.

[0023] As used herein, the term oligonucleotide is defined as a molecule that is generally understood by those skilled in the art to contain two or more covalently linked nucleotides. For use as oligonucleotides with therapeutic value, oligonucleotides are typically synthesized to have a length of 10 to 40 nucleotides, preferably 10 to 25 nucleotides.

[0024] Oligonucleotides may include optionally modified DNA or RNA nucleoside monomers or combinations thereof.

[0025] As used herein, optional modification refers to a nucleoside modified by introducing one or more modifications to the sugar or nucleobase moiety compared to an equivalent DNA or RNA nucleoside.

[0026] Typical modifications can be 2'-O-(2-methoxyethyl)-substitution (2'-MOE) in the sugar moiety or locked RNA (LNA), which is a modified RNA nucleotide in which the ribose moiety is modified by an additional bridge connecting the 2' oxygen and the 4' carbon.

[0027] The term "modified nucleoside" may also be used interchangeably with the terms "nucleoside analogue," "modified unit," or "modified monomer" in this document.

[0028] DNA or RNA nucleotides are typically linked by phosphodiester (P=O) or thiophosphate (P=S) nucleotides that covalently couple two nucleotides together.

[0029] According to the present invention, at least one internucleotide bond must consist of a phosphate thioester (P=S). Therefore, in some oligonucleotides, all other internucleotide bonds may consist of a phosphate diester (P=O), or in other oligonucleotides, the internucleotide bond sequences are different and include both phosphate diester (P=O) and phosphate thioester (P=S) internucleotide bonds.

[0030] Therefore, the term mixed P=O / P=S backbone oligonucleotide refers to an oligonucleotide in which at least one nucleotide bond must consist of a phosphate thioester (P=S) and at least one nucleotide bond must consist of a phosphate diester (P=O).

[0031] The nucleobase moiety can be represented by a letter code for each corresponding nucleobase, such as A, T, G, C, or U, where each letter may optionally include a modified nucleobase with equivalent function. For example, in the example oligonucleotide, for the LNA nucleoside, the nucleobase moiety is represented by uppercase letters A, T, G, and U. Me C(5-methylcytosine) is used to describe the nucleobase moiety of DNA nucleosides, where the nucleobases are represented by lowercase letters a, t, g, c, and t. Me Description C. Modified nucleobases include, but are not limited to, nucleobases with 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).

[0032] Preferably, the oligonucleotide comprises optionally modified DNA or RNA nucleoside monomers or combinations thereof, having a length of 10 to 40, preferably 10 to 25 nucleotides.

[0033] The principles of oligonucleotide synthesis are well known in the field (see, for example, oligonucleotide synthesis; Wikipedia, the free encyclopedia; https: / / en.wikipedia.org / wiki / Oligonucleotide synthesis, March 15, 2016).

[0034] Today, larger-scale oligonucleotide synthesis is carried out in an automated manner using computer-controlled synthesizers.

[0035] Oligonucleotide synthesis is typically a solid-phase synthesis, in which the oligonucleotide being assembled is covalently bound to a solid support material via its 3'-terminal hydroxyl group and remains attached to it throughout the chain assembly process. Suitable supports are commercially available macroporous polystyrene supports, such as GE Healthcare's Primer support 5G or Kinovate. HL support.

[0036] Subsequent cleavage from the resin can be carried out using concentrated ammonia. Protecting groups on phosphate esters and nucleotide bases are also removed during this cleavage process.

[0037] As outlined above, a method for producing mixed P=O / P=S backbone oligonucleotides includes oxidizing an intermediate phosphite compound of formula I into a phosphodiester compound of formula II using an oxidizing solution containing iodine, an organic solvent, and water.

[0038] An oxidizing solution can be prepared by mixing an iodide with water and an organic solvent and then adding iodine.

[0039] The iodide is selected from hydrogen iodide, basic iodides, or basic triiodides, preferably from hydrogen iodide or basic iodides, and more preferably from sodium iodide or potassium iodide.

[0040] The organic solvent can be selected from pyridine or C. 1-6 Alkyl-substituted pyridines, such as dimethylpyridine, but preferably derived from pyridine. Other organic solvents, such as tetrahydrofuran, may be present.

[0041] The volume ratio of organic solvent to water is selected from 1:1 to 20:1, preferably from 5:1 to 15:1, and more preferably 9:1.

[0042] 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, preferably 1.0:1.0 to 1.0:2.0.

[0043] The iodine concentration in the oxidizing solution is typically in the range of 10 mM to 100 mM, preferably in the range of 15 mM to 60 mM.

[0044] Based on an iodine content of 50 mM, a certain amount of iodide is added until the oxidized solution has a conductivity of ≥1500 μS / cm.

[0045] In a preferred embodiment, the iodide is potassium iodide and the oxidizing solution has a conductivity of ≥1500 μS / cm, preferably between 1650 μS / cm and 2050 μS / cm, and more preferably between 1750 μS / cm and 1950 μS / cm, based on a content of 50 mM KI and 50 mM I2.

[0046] Based on an iodine content of 10 mM, a certain amount of iodide is added until the oxidized solution has a conductivity of ≥300 μS / cm.

[0047] In a preferred embodiment, the iodide is potassium iodide and the oxidizing solution has a conductivity of ≥300 μS / cm, preferably between 350 μS / cm and 550 μS / cm, and more preferably between 400 μS / cm and 500 μS / cm, based on 10 mM KI and 10 mM I2.

[0048] Based on an iodine content of 20 mM, a certain amount of iodide is added until the oxidized solution has a conductivity of ≥600 μS / cm.

[0049] In a preferred embodiment, the iodide is potassium iodide and the oxidizing solution has a conductivity of ≥600 μS / cm, preferably between 750 μS / cm and 950 μS / cm, and more preferably between 800 μS / cm and 900 μS / cm, based on 20 mM KI and 20 mM I2.

[0050] Based on an iodine content of 100 mM, a certain amount of iodide is added until the oxidized solution has a conductivity of ≥3000 μS / cm.

[0051] In a preferred embodiment, the iodide is potassium iodide and the oxidizing solution has a conductivity of ≥3000 μS / cm, preferably between 3200 μS / cm and 3900 μS / cm, and more preferably between 3350 μS / cm and 3750 μS / cm, based on 100 mM KI and 100 mM I2.

[0052] Typically, an oxidizing solution can oxidize the intermediate triphosphite compound of Formula I to the phosphodiester compound of Formula II in such a way that the P=O content in the reaction solution reaches a value of less than 2.5%, preferably less than 2.0%.

[0053] As a further embodiment of the present invention, a method for evaluating the quality of an oxidizing solution is provided, comprising:

[0054] a) Provide an oxidizing solution containing iodine, an organic solvent, and water.

[0055] b) Measure the conductivity of the oxidizing solution, and

[0056] c) Assess the suitability of the oxidizing solution for oxidizing the intermediate triphosphite compound of Formula I to the phosphodiester compound of Formula II based on a certain threshold of the measured conductivity.

[0057] As a further, more preferred embodiment of the method for evaluating the quality of an oxidizing solution, the oxidizing solution additionally contains iodide.

[0058] The amount of iodine used in the preparation of the oxidation reaction is typically selected between 1.1 and 15 equivalents, more preferably between 1.5 and 4.5 equivalents.

[0059] The oxidation reaction is carried out between 15°C and 27°C, more preferably between 18°C ​​and 24°C.

[0060] As summarized above, in a preferred embodiment of the invention, the oxidizing solution can be applied immediately after its preparation, based on the stoichiometric ratio of iodine to iodide or the ratio of excess iodide present.

[0061] However, in another, less preferred embodiment of the invention, the ratio of iodine and iodide to a substoichiometric amount of iodide can be used.

[0062] Such oxidizing solutions may require a certain amount of time to mature until they possess the desired properties, in terms of conductivity and the potential to selectively oxidize the phosphite triester compound of formula I to the phosphodiester compound of formula II.

[0063] The optimal aging time depends largely on the aging temperature of the oxidizing solution. Lower aging temperatures result in longer aging times, while higher aging temperatures significantly shorten the aging time.

[0064] For example, the oxidizing solution can be aged at a temperature of 20°C to 100°C, but preferably at a temperature of 30°C to 60°C.

[0065] The time required for aging in the oxidative solution must be sufficient to selectively oxidize the phosphite compound of Formula I to the phosphodiester compound of Formula II without affecting the internucleotide bonding of the thiophosphate.

[0066] Typically, the aging period for an oxidizing solution is at least 1 day, 3 days, 5 days, 10 days, 15 days, or at least 20 days.

[0067] As mentioned above, the time period can vary greatly depending on the aging temperature, and for aging temperatures of 30°C to 35°C, it can vary between 10 days and 150 days, more typically between 20 days and 60 days, while for aging temperatures of 60°C to 65°C, it can vary between 1 day and 30 days, typically between 2 days and 15 days.

[0068] Maturation is typically accompanied by an increase in conductivity (μS / cm) and a decrease in pH until a plateau is reached.

[0069] In another embodiment, the invention includes a novel oxidation solution that may comprise:

[0070] a) 10mM to 100mM iodine

[0071] b) Iodides ranging from 0.1 mol to 3.0 mol equivalents associated with 1.0 mol equivalent of iodine.

[0072] c) Organic solvents, and

[0073] d) Water, wherein the volume ratio of organic solvent to water is 20:1 to 1:1.

[0074] Preferably,

[0075] a) 15mM to 60mM iodine

[0076] b) Iodides of 1.0 mol to 2.0 mol equivalents associated with 1.0 mol equivalent of iodine.

[0077] c) Organic solvents, and

[0078] d) Water, wherein the volume ratio of organic solvent to water is 5:1 to 15:1.

[0079] More preferably,

[0080] a) 15mM to 60mM iodine

[0081] b) 1.0 mol to 2.0 mol equivalents of hydrogen iodide or basic iodide associated with 1.0 mol equivalent of iodine.

[0082] c) Pyridine, and

[0083] d) Water, wherein the volume ratio of pyridine to water is 5:1 to 15:1.

[0084] Even more preferably,

[0085] a) 15mM to 60mM iodine

[0086] b) 1.0 mol to 2.0 mol equivalents of sodium iodide or potassium iodide associated with 1.0 mol equivalent of iodine.

[0087] c) Pyridine, and

[0088] d) Water, wherein the volume ratio of pyridine to water is 9:1.

[0089] For example, oligonucleotides can be selected from:

[0090] 5′- Me C S Me U O Me C O A O G S T S A S A S Me C S A S T S T S G S A S Me C S A O Me C O Me C O A S Me C -

[0091] 3′

[0092] The underlined residues are 2'-MOE nucleosides. The positions of the thiophosphate and phosphate diester bonds are indicated by S and O, respectively. It should be noted that 2'-O-(2-methoxyethyl)-5-methyluridine (2'-MOE MeU) nucleoside is sometimes referred to as 2'-O-(2-methoxyethyl) nucleoside (2'-MOE T).

[0093] The compounds disclosed herein have the following nucleobase sequence

[0094] SEQ ID No.1:cucagtaacattgacaccac

[0095] Example

[0096] synthesis

[0097] 5′-Me C S Me U O Me C O A O G S T S A S A S Me C S A S T S T S G S A S Me C S A O Me C O Me C O A S Me C -3′

[0098] Oligonucleotides were produced on a solid-phase scale at a scale of 2.20 mmol using the standard phosphoramidite chemistry method with AKTA Oligopilot 100 and Primer SupportUnylinker (NittoPhase LH Unylinker 330). Typically, 1.4 equivalents of DNA / MOE-phosphoramidite were used. Other reagents (dichloroacetic acid, 1-methylimidazole, 4,5-dicyanimidazole, acetic anhydride, phenylacetyl disulfide, pyridine, triethylamine) were used as is from commercially available sources, and reagent solutions of appropriate concentrations were prepared (see below for details). The oxidizing agent solution was freshly prepared (see below). Cleavage and deprotection were achieved using ammonium hydroxide to obtain crude oligonucleotides.

[0099] Standard reagent solution

[0100] Unlocked A 10% dichloroacetic acid solution in toluene (v / v) phosphorus amide 0.2M acetonitrile solution NMI / DCI activator 1.0M 4,5-dicyanimidazolium / 0.1M 1-methylimidazolium acetonitrile solution Thiolation 0.2M phenylacetyl disulfide 3-methylpyridine / acetonitrile solution (1:1 v / v) End cap A 1-Methylimidazolium / pyridine / acetonitrile 2:3:5 (v / v / v) End cap B Acetic anhydride / acetonitrile 1:4 (v / v) Amine washing solution 50% triethylamine in acetonitrile solution (v / v) Pyrolysis and Deprotection 28–32% ammonium hydroxide aqueous solution

[0101] Preparation of iodine / potassium iodide solution

[0102] Add potassium iodide to water at room temperature, then add pyridine. Add iodine and stir the mixture under positive pressure of dry nitrogen for 1 hour before use.

[0103]

[0104] Preparation of iodine / sodium iodide solution

[0105] Add 7.49 g of sodium iodide to 101 g of water at room temperature, then add 886 g of pyridine. Add 12.7 g of iodine and stir the mixture under positive pressure of dry nitrogen for 1 hour before use.

[0106] Oxidation examples using different oxidant solutions without aging

[0107]

[0108] 1 It refers to the percentage of molecules with a mass difference of 16 Da relative to the molecular mass of the desired compound determined in mass spectrometry, that is, the percentage of molecules in which one P=S bond has been converted to a P=O bond.

[0109] Aging of KI (50 mM) / I2 (50 mM) solution at 30°C to 35°C

[0110] The solution should be stored in amber glass bottles at 30°C to 35°C until use.

[0111] Oxidation example using a conditioned (30°C to 35°C) KI (50mM) / I2 (50mM) solution.

[0112]

[0113]

[0114] 1 It refers to the percentage of molecules with a mass difference of 16 Da relative to the molecular mass of the desired compound determined in mass spectrometry, that is, the percentage of molecules in which one P=S bond has been converted to a P=O bond.

Claims

1. A method for producing mixed P=O / P=S backbone oligonucleotides, comprising oxidizing an intermediate phosphite compound of formula I into a phosphodiester compound of formula II using an oxidizing solution containing iodine, an organic solvent, and water, according to a scheme. Its features The oxidizing solution additionally contains iodide, and The oxidizing solution is aged at a temperature of 30°C to 35°C for 10 to 150 days.

2. The method according to claim 1, wherein the iodide is selected from hydrogen iodide, basic iodides, or basic triiodides.

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

4. The method according to claim 1, wherein the iodide is selected from basic iodides.

5. The method according to claim 1, 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 according to any one of claims 1 to 5, 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 method according to claim 1, wherein the organic solvent is selected from pyridine or C 1-6 Alkyl-substituted pyridine.

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

9. The method according to any one of claims 1 to 5 and 7 to 8, wherein the volume ratio of organic solvent to water is from 1:1 to 20:

1.

10. The method of claim 9, wherein the volume ratio of the organic solvent to water is from 5:1 to 15:

1.

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

1.

12. The method according to any one of claims 1 to 5, 7 to 8 and 10 to 11, wherein the iodine concentration in the oxidizing solution is from 10 mM to 100 mM.

13. The method of claim 12, wherein the iodine concentration in the oxidizing solution is from 15 mM to 60 mM.

14. The method according to any one of claims 1 to 5, 7 to 8, 10 to 11 and 13, wherein the oxidizing solution used has an iodine content of 50 mM and iodide has been added to the oxidizing solution until the oxidizing solution has a conductivity of ≥1500 μS / cm.

15. The method of claim 14, wherein the oxidizing solution used has a conductivity of ≥1500 μS / cm based on a content of 50 mM KI and 50 mM I2.

16. The method of claim 15, wherein the oxidizing solution used has a conductivity between 1650 μS / cm and 2050 μS / cm based on a content of 50 mM KI and 50 mM I2.

17. The method according to any one of claims 1 to 5, 7 to 8, 10 to 11 and 13, wherein the oxidizing solution used has an iodine content of 10 mM, and iodide has been added to the oxidizing solution until the oxidizing solution has a conductivity of ≥300 μS / cm.

18. The method of claim 17, wherein the oxidizing solution used has a conductivity of ≥300 μS / cm based on a content of 10 mM KI and 10 mM I2.

19. The method of claim 18, wherein the oxidizing solution used has a conductivity between 350 μS / cm and 550 μS / cm based on a content of 10 mM KI and 10 mM I2.

20. The method according to any one of claims 1 to 5, 7 to 8, 10 to 11 and 13, wherein the oxidizing solution used has an iodine content of 20 mM, and iodide has been added to the oxidizing solution until the oxidizing solution has a conductivity of ≥600 μS / cm.

21. The method of claim 20, wherein the oxidizing solution used has a conductivity of ≥600 μS / cm based on a content of 20 mM KI and 20 mM I2.

22. The method of claim 21, wherein the oxidizing solution used has a conductivity between 750 μS / cm and 950 μS / cm based on a content of 20 mM KI and 20 mM I2.

23. The method according to any one of claims 1 to 5, 7 to 8, 10 to 11 and 13, wherein the oxidizing solution used has an iodine content of 100 mM, and an iodide has been added to the oxidizing solution until the oxidizing solution has a conductivity of ≥3000 μS / cm.

24. The method of claim 23, wherein the oxidizing solution used has a conductivity of ≥3000 μS / cm based on a content of 100 mM KI and 100 mM I2.

25. The method of claim 24, wherein the oxidizing solution used has a conductivity between 3200 μS / cm and 3900 μS / cm based on a content of 100 mM KI and 100 mM I2.

26. The method according to any one of claims 1 to 5, 7 to 8, 10 to 11, 13, 15 to 16, 18 to 19, 21 to 22 and 24 to 25, wherein the oxidizing solution is capable of oxidizing the intermediate triphosphite compound of formula I to the phosphodiester compound of formula II in such a way that the P=O content in the reaction solution reaches a value of less than 2.5%.

27. The method according to claim 26, wherein the oxidizing solution is capable of oxidizing the intermediate triphosphite compound of formula I to the phosphodiester compound of formula II in such a way that the P=O content in the reaction solution reaches a value of less than 2.0%.

28. The method according to any one of claims 1 to 5, 7 to 8, 10 to 11, 13, 15 to 16, 18 to 19, 21 to 22, 24 to 25 and 27, wherein the amount of iodine used to prepare the oxidizing solution is selected between 1.1 equivalents and 15 equivalents.

29. The method of claim 28, wherein the amount of iodine used to prepare the oxidation solution is selected between 1.5 equivalents and 4.5 equivalents.

30. The method according to any one of claims 1 to 5, 7 to 8, 10 to 11, 13, 15 to 16, 18 to 19, 21 to 22, 24 to 25, 27 and 29, wherein the reaction temperature of the oxidation reaction is selected between 15°C and 27°C.

31. The method according to claim 30, wherein the reaction temperature of the oxidation reaction is selected between 18°C ​​and 24°C.

32. The method according to any one of claims 1 to 5, 7 to 8, 10 to 11, 13, 15 to 16, 18 to 19, 21 to 22, 24 to 25, 27, 29 and 31, wherein the oligonucleotide comprises optionally modified DNA or RNA nucleoside monomers or combinations thereof, and has a length of 10 to 40 nucleotides.

33. The method of claim 32, wherein the oligonucleotide comprises optionally modified DNA or RNA nucleoside monomers or combinations thereof, and has a length of 10 to 25 nucleotides.

34. An oxidizing solution comprising a) 10mM to 100mM iodine b) Iodides ranging from 0.1 mol to 3.0 mol equivalents associated with 1.0 mol equivalent of iodine. c) Organic solvents, and d) Water, wherein the volume ratio of organic solvent to water is 20:1 to 1:1, and The oxidizing solution is aged at a temperature of 30°C to 35°C for 10 to 150 days.

35. The oxidizing solution according to claim 34, comprising... a) 15mM to 60mM iodine b) Iodides of 1.0 mol to 2.0 mol equivalents associated with 1.0 mol equivalent of iodine. c) Organic solvents, and d) Water, wherein the volume ratio of the organic solvent to water is from 5:1 to 15:

1.

36. The oxidizing solution according to claim 34, comprising: a) 15mM to 60mM iodine b) 1.0 mol to 2.0 mol equivalents of hydrogen iodide or basic iodide associated with 1.0 mol equivalent of iodine. c) Pyridine, and d) Water, wherein the volume ratio of the organic solvent to water is from 5:1 to 15:1.

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