Method for preparing oligonucleotides using a modified oxidation protocol

Abstract

To provide a method for producing mixed P=O / P=S skeleton oligonucleotide.SOLUTION: A method includes selective oxidation of an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II according to a scheme using oxidation solution obtained by mixing iodine, organic solvent and water, where the oxidation solution is aged for a sufficient time in order to selectively oxidize the phosphite triester compound of formula I into the phosphodiester compound of formula II without oxidizing inter phosphorothioate nucleotide bond.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides a novel method for preparing mixed P=O / P=S backbone oligonucleotides, comprising the steps of: oxidizing an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II according to TIFF0007798962000001.tif47147; wherein said oxidation follows a specific oxidation protocol. [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: a1) deblocking protected hydroxyl groups on the solid support; a2) coupling a first nucleoside as an activated phosphoramidite to a free hydroxyl group on said 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 said respective P-linked dinucleotides (phosphite triesters) to form respective phosphodiesters (P=O) or 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 principles of oligonucleotide synthesis are well known in the art (see, for example, Wikipedia, the free encyclopedia, Oligonucleotide synthesis, https: / / en.wikipedia.org / wiki / Oligonucleotide synthesis, March 15, 2016 (Non-Patent Document 1)).

[0005] The oxidation step is typically carried out using an oxidation solution containing iodine, an organic solvent, generally pyridine, and water.

[0006] 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 also, 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. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Oligonucleotide synthesis, https: / / en.wikipedia.org / wiki / Oligonucleotide synthesis, March 15, 2016 Summary of the Invention

[0008] 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.

[0009] The object of the present invention is a method for preparing a mixed P=O / P=S backbone oligonucleotide, comprising the steps of: oxidizing an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II, Scheme using an oxidizing solution obtained by mixing iodine, an organic solvent and water: It has been found that this can be achieved by a method comprising: performing in accordance with TIFF0007798962000002.tif47147, wherein the oxidizing solution is aged for a time sufficient to selectively oxidize the phosphite triester compound of formula I to the phosphodiester compound of formula II without oxidizing the phosphorothioate internucleotide linkages.

[0010] [The present invention 1001] 1. A method for producing a mixed P=O / P=S backbone oligonucleotide, comprising: Scheme using an oxidizing solution obtained by mixing iodine, an organic solvent and water: TIFF0007798962000003.tif47147, which comprises the oxidation of an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II, wherein the oxidizing solution is aged for a time sufficient to selectively oxidize the phosphite triester compounds of formula I to the phosphodiester compounds of formula II without oxidizing phosphorothioate internucleotide linkages. [The present invention 1002] The organic solvent is pyridine or C 1-6 1001. The method of claim 1001, wherein the compound is an alkyl-substituted pyridine. [The present invention 1003] Pyridine or C 1-6The process according to claim 1002, wherein the volume ratio of alkyl-substituted pyridine to water is 1:1 to 20:1, preferably 5:1 to 15:1, more preferably 9:1. [The present invention 1004] The method according to claim 1002 or 1003, wherein the concentration of said iodine in said oxidizing solution is 10 mM to 100 mM, more preferably 20 mM to 50 mM. [The present invention 1005] 1005. The process according to any one of claims 1001 to 1004, wherein said aging of said oxidizing solution is carried out at a temperature between 20°C and 100°C, preferably between 30°C and 60°C. [The present invention 1006] 1006. The method of any of claims 1001 to 1005, wherein the oxidizing solution has been aged for at least 1 day, 3 days, 5 days, 10 days, 15 days, or at least 20 days. [The present invention 1007] 1006. The method of any of claims 1001 to 1006, comprising monitoring the pH and conductivity to determine a time sufficient to selectively oxidize the phosphite triester compound of formula I to the phosphodiester compound of formula II without oxidizing the phosphorothioate internucleotide linkages. [The present invention 1008] The method of any one of claims 1001 to 1007, wherein the amount of oxidizing agent used in the oxidation reaction is selected from the range of 1.1 equivalents to 15 equivalents, more preferably 1.5 equivalents to 4.5 equivalents, and most preferably 2 equivalents to 4 equivalents. [The present invention 1009] 1009. The process of any one of claims 1001 to 1008, wherein the reaction temperature for said oxidation reaction is selected between 15°C and 27°C, more preferably between 18°C ​​and 24°C. [The present invention 1010] 1009. The method of any of claims 1001 to 1009, wherein said 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. The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe this invention.

[0011] "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.

[0012] 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.

[0013] Oligonucleotides can be composed of optionally modified DNA or RNA nucleoside monomers or combinations thereof.

[0014] 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.

[0015] 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.

[0016] The term modified nucleoside may be used interchangeably with the terms "nucleoside analogue" or modified "unit" or modified "monomer."

[0017] 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.

[0018] 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.

[0019] 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).

[0020] 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 for DNA nucleosides lowercase a, t, g, c and M Modified nucleobases include, but are not limited to, nucleobases bearing protecting groups such as tert-butylphenoxyacetyl, phenoxyacetyl, benzoyl, acetyl, isobutyryl, or dimethylformamidino (see Wikipedia's Phosphoramidit-Synthese, https: / / de.wikipedia.org / wiki / Phosphoramidit-Synthese, March 24, 2016).

[0021] 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.

[0022] 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).

[0023] Today, large scale oligonucleotide synthesis is performed in an automated manner using computer-controlled synthesizers.

[0024] Generally, oligonucleotide synthesis is a solid-phase synthesis, in which the oligonucleotide to be assembled is covalently attached to a solid support material via its 3'-terminal hydroxy group and remains 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.

[0025] 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.

[0026] As outlined above, the process for preparing 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: A scheme using an oxidizing solution obtained by mixing iodine, an organic solvent, and water: TIFF0007798962000004.tif47147, wherein the oxidizing solution is aged for a time sufficient to selectively oxidize the phosphite triester compound of Formula I to the phosphodiester compound of Formula II without oxidizing the phosphorothioate internucleotide linkages.

[0027] A mixed P=O / P=S backbone oligonucleotide contains at least one phosphorothioate internucleotide linkage.

[0028] The oxidizing solution is typically a solution obtained by mixing iodine, an organic solvent, and water.

[0029] The organic solvent is pyridine or C 1-6 It may be selected from alkyl substituted pyridines such as lutidine, but preferably pyridine.An additional organic solvent such as tetrahydrofuran may be present.

[0030] Oxidizing solutions are commercially available, for example, from Sigma Aldrich (Merck) as oxidizer solution. Alternatively, a fresh solution can be prepared using commercially available iodine and pyridine.

[0031] Pyridine or C 1-6 The volume ratio of alkyl-substituted pyridine to water can vary within the range of 1:1 to 20:1, preferably 5:1 to 15:1, but is more preferably 9:1.

[0032] The iodine concentration in the oxidizing solution can be in the range of 10 mM to 100 mM, more preferably in the range of 20 mM to 50 mM.

[0033] 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.

[0034] It has been found that the aging of the oxidizing solution can be carried out at a temperature between 20°C and 100°C, but preferably between 30°C and 60°C.

[0035] 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 oxidizing the phosphorothioate internucleotide linkages.

[0036] 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.

[0037] 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 between 10 and 150 days, more typically between 20 and 60 days, while at aging temperatures of 60°C to 65°C, it can vary between 1 and 30 days, more typically between 2 and 15 days.

[0038] Aging is generally accompanied by an increase in conductivity (μS / cm) and a decrease in pH. In a further embodiment of the invention, the method includes monitoring the parameters pH and conductivity to determine a time sufficient to selectively oxidize the phosphite triester compound of formula I to the phosphodiester compound of formula II without oxidizing the phosphorothioate internucleotide linkages.

[0039] The amount of the oxidizing agent used in the oxidation reaction can be selected from the range of 1.1 to 15 equivalents, more preferably 1.5 to 4.5 equivalents, and most preferably 2 to 4 equivalents.

[0040] Generally, the oxidation reaction temperature is between 15°C and 27°C, and more preferably between 18°C ​​and 24°C.

[0041] By way of example, the oligonucleotide may be selected from: 5'- MeC 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' 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).

[0042] The compounds disclosed herein have the following nucleobase sequences: SEQ ID NO: 1: cucagtaacattgacaccac [Example]

[0043] Example 1 5'- Me C S Me U O Me C O AO 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 Synthesis of -3' Oligonucleotides were prepared on a 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 / 2'-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 Table 1 below). Cleavage and deprotection were achieved using ammonium hydroxide to yield the crude oligonucleotides.

[0044] (Table 1) Standard reagent solutions TIFF0007798962000005.tif66169

[0045] Example 2 Oxidant aging experiment Example 2.1 Use a purchased oxidizer solution

[0046] (Table 2) TIFF0007798962000006.tif531601 refers to the time when an aliquot from the commercial solution was taken for use testing and heat treatment of the remainder of the solution began. This is not the same as the time of preparation of the solution. 2 The solutions were not aged at 30-35°C and were stored at 1-15°C starting from t=0. 3 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.

[0047] Example 2.2 Use a freshly prepared oxidizer solution, a) Preparation of iodine solution 1.00 kg of water was added to 8.00 kg of pyridine at room temperature. 127 g of iodine was added. 0.827 kg of pyridine was added for rinsing, and the mixture was stirred under a positive pressure of dry nitrogen for 1 hour. b) Aging of iodine solution Aging at 30-35°C, · 800 mL aliquots were stored in amber glass bottles at 30-35°C until use. Aging at 60-65°C: The material was kept at 60–65 °C in a jacketed glass reactor under a positive pressure of dry nitrogen until use.

[0048] (Table 3) Aging at 30℃-35℃ TIFF0007798962000007.tif58128

[0049] (Table 4) Aging at 60℃-65℃ TIFF0007798962000008.tif52128 1 refers to the point at which the solution is ready. 2refers 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.

[0050] Sequence information SEQUENCE LISTING <110> F. Hoffmann-La Roche AG <120> Process for the preparation of oligonucleotides using modified oxidation protocol <150> EP 19179310.8 <151> 2019-06-11 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Oligonucleotide <400> 1 cucagtaaca ttgacaccac 20

Claims

1. 1. A method for producing a mixed P=O / P=S backbone oligonucleotide, comprising: Scheme using an oxidizing solution obtained by mixing iodine, pyridine and water: The method comprises the oxidation of an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II, according to wherein the oxidation solution has been aged at a temperature of 30° C. to 60° C. for at least 3 days, 5 days, 10 days, 15 days, or at least 20 days to selectively oxidize the phosphite triester compound of Formula I to the phosphodiester compound of Formula II without oxidizing the phosphorothioate internucleotide linkages.

2. 2. The method of claim 1, wherein the volume ratio of pyridine to water is from 1:1 to 20:

1.

3. 3. The method of claim 1, wherein the concentration of the iodine in the oxidizing solution is 10 mM to 100 mM.

4. 4. The method of claim 1, comprising monitoring the pH and conductivity to determine the time required for aging the oxidizing solution to selectively oxidize the phosphite triester compound of formula I to the phosphodiester compound of formula II without oxidizing the phosphorothioate internucleotide linkages.

5. 5. The method according to any one of claims 1 to 4, wherein the amount of oxidizing agent used in the oxidation reaction is selected between 1.1 equivalents and 15 equivalents.

6. 6. The method according to any one of claims 1 to 5, wherein the reaction temperature for the oxidation reaction is selected between 15°C and 27°C.

7. 7. The method of any one of claims 1 to 6, wherein the oligonucleotide is composed of optionally modified DNA or RNA nucleoside monomers or a combination thereof and is 10 to 40 nucleotides in length.

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