Production of oligonucleic acid

Abstract

The purpose of the present invention is to provide a method for efficiently producing an oligonucleic acid having a mixed P=O / P=S skeleton, with which it becomes possible to suppress the production of a PS-OP variant that is unexpectedly produced as a byproduct in the production of the oligonucleic acid having a mixed P=O / P=S skeleton.　The present invention relates to a method for producing an oligonucleic acid having a mixed P=O / P=S skeleton, the method being characterized in that an oxidation solution contains iodine, an organic solvent, and water and is additionally contains a trivalent phosphorus compound represented by the formula [wherein R1, R2, and R3 may be the same as or different from one another and each independently represent an optionally substituted C6-14 aryl or C1-6 alkyl group, wherein two R moieties may be bonded to each other and bonded to another adjacent atom to form a ring] at a concentration of 100-500 mM.

Description

Production of oligonucleotides

[0001] This patent application claims priority and interest under the Paris Convention and priority under Article 41 of the Japanese Patent Act, based on Japanese Patent Application No. 2024-192214 (filed on 31 October 2024), and by reference herein the entire contents of the said application are incorporated herein by reference.

[0002] The present invention relates to a method for producing oligonucleotides. More specifically, the present invention relates to a method for producing mixed P=O / P=S backbone oligonucleotides, wherein the oxidizing solution contains iodine, an organic solvent, and water, and the oxidizing solution further contains a specific trivalent phosphorus compound at a specific concentration, and to an oxidizing solution used in the method.

[0003] Oligonucleotides can be manufactured using either solid-phase synthesis or liquid-phase synthesis. Liquid-phase synthesis requires extraction and purification at each step, such as deprotection and bond formation, resulting in a long time and considerable effort. On the other hand, in solid-phase synthesis, all reactions proceed on beads, and excess reagents and by-products can be easily removed by washing. For these reasons, solid-phase synthesis eliminates the need for complicated operations, allows for rapid and efficient acquisition of the target product, and is also automatable. Consequently, solid-phase synthesis has become increasingly popular for the production of oligonucleotides in recent years.

[0004] Oligonucleotides such as DNA and RNA have a structure in which many nucleic acid units are linked together by phosphate ester bonds. This phosphate ester bond is rarely found in other small molecule compounds and requires a unique bond formation reaction. In nucleic acid synthesis, this bond formation reaction is repeated many times, so its efficiency greatly affects the overall yield.

[0005] In solid-phase synthesis of oligonucleotides, the phosphoramidite method, which uses phosphoramidites of nucleosides, is the mainstream method. The phosphoramidite method for solid-phase synthesis of oligonucleotides includes the following steps: (1) deprotecting the protecting group on the support; (2) coupling the phosphoramidite; (3) oxidizing the resulting phosphite ester; (4) capping the unreacted hydroxyl group by acetylating it; (5) repeating steps (1) to (4) to extend the chain as needed; and (6) deprotecting the obtained oligonucleotide and cleaving it from the support. The principle of oligonucleotide synthesis is well known in this industry (for example, Non-Patent Document 1).

[0006] In recent years, nucleic acid drugs, which utilize oligonucleotides as medicines, have been rapidly gaining attention. However, a problem with nucleic acid drugs is that phosphate ester bonds within the oligonucleotides can be cleaved by nucleases in the human body, causing the nucleic acid molecules to be degraded before reaching the affected area. Therefore, it is common practice to convert the phosphate ester bonds, which are targeted by enzymes, into thiophosphate ester bonds (phosphorothioates). Oligonucleotides with phosphorothioate bonds have the characteristics of being resistant to nucleases, yet can undergo hybridization with antisense strands without problems, and can form Watson-Crick base pairs. Furthermore, the synthesis of phosphorothioates involves almost the same steps as the solid-phase synthesis method of oligonucleotides using the phosphoramidite method, making it simple and low-cost.

[0007] In the solid-phase synthesis method, the step of oxidizing the generated phosphite ester (3) is typically carried out using an oxidation solution containing iodine, an organic solvent which is generally pyridine, and water. However, in the production of a mixed P=O / P=S backbone oligonucleic acid having both a phosphoester bond and a phosphorothioate bond in the nucleotide bond in an oligonucleic acid, when applying a conventional oxidation solution, not only does the desired oxidation of the triester phosphite moiety of the following formula (X) to the phosphodiester moiety of formula (Y) occur, but as a side reaction, the phosphorothioate bond present in the molecule is converted to a phosphoester bond, that is, P=S is converted to P=O in the nucleotide bond, and unexpectedly, a PS-PO variant is by-produced with respect to the desired mixed P=O / P=S backbone oligonucleic acid, and it has been observed that the content of the by-produced mixed P=O / P=S backbone oligonucleic acid can be higher than expected.

[0008]

[0009] To solve this inconvenience, in Patent Document 1, the production of the PS-PO variant is suppressed by aging a newly prepared oxidation solution for a long period or by adding an iodide. In Patent Document 2, the production of the PS-PO variant is suppressed by aging a newly prepared oxidation solution for a long period, and in Patent Document 3, it is described that the production of the PS-PO variant without aging is suppressed by adding an iodide to a newly prepared oxidation solution.

[0010] However, for any suppression of the production of the PS-PO variant, in the case of aging, it is necessary to specialize the production equipment, and with the iodide addition amounts described in the patent documents, there are cases where a sufficient effect cannot be obtained depending on the sequence of the target oligonucleic acid and the oxidation conditions, and the iodides used are unstable to air and light, and many of them are hygroscopic compounds, and it is easy for variations to occur in the iodide concentration in the solution each time production is carried out, etc., and each has problems to be solved.

[0011] WO2020-236618 Gazette, Special Table 2022-536157 Gazette, Special Table 2023-533017 Gazette, WO2020-196890 Gazette

[0012] Wikipedia's Oligonucleotide Synthesis, Https: / / en.wikipedia.org / wiki / Oligonucleotide synthesis, March 15, 2016.

[0013] In the production of mixed P=O / P=S backbone oligonucleic acids, there is still a need for an efficient production method that suppresses the generation of PS-PO variants, which are unexpectedly by-produced, as much as possible.

[0014] The inventors of the present invention conducted intensive studies on the oxidation solution used in the above oxidation reaction, and found that by adding a trivalent phosphorus compound represented by iodine and triphenylphosphine to the oxidation solution, the generation of PS-PO variants can be efficiently suppressed, and thus completed the present invention.

[0015] That is, the present invention includes the following aspects. <Production method characterized by a trivalent phosphorus compound> [1] A method for producing a mixed P=O / P=S backbone oligonucleic acid, comprising using an oxidation solution containing iodine, an organic solvent, preferably pyridine or C １－６ alkyl-substituted pyridine, and water, and oxidizing the phosphite triester moiety of formula (I):

[0016] [wherein, R １１ represents a C １－６ alkyl group optionally substituted with CN, preferably cyanoethyl, B each independently represents a nucleobase optionally protected with a protecting group, and R each independently represents a protected hydroxyl group, a hydrogen atom, a fluorine atom, a methoxy group, or a 2-methoxyethyl group] to the phosphodiester moiety of formula (II), and the oxidation solution further has the formula:

[0017] [wherein, R １ , R ２ and R ３ may be the same or different and each independently represents an optionally substituted C ６－１４ aryl group or C １－６The method is characterized by containing a trivalent phosphorus compound represented by [an alkyl group, in which two Rs are bonded to each other and may bond to other adjacent atoms to form a ring] in an oxidation solution at a concentration of 100 mM to 500 mM.

[0018] [2] The method according to [1], wherein the oxidizing solution selectively oxidizes the phosphite triester portion of formula (I) to the phosphodiester portion of formula (II) without oxidizing the phosphorothioate nucleotide bonds in the mixed P=O / P=S backbone oligonucleotide. [3-1] The method according to [1] or [2], wherein the trivalent phosphorus compound is contained in the oxidizing solution at a concentration of 100 mM to 500 mM. [3-2] The method according to [3-1], wherein the trivalent phosphorus compound is contained in the oxidizing solution at a concentration of 100 mM to 300 mM. [3-3] The method according to [3-2], wherein the concentration of the trivalent phosphorus compound is 100 mM to 250 mM in the oxidizing solution. [3-4] The method according to [3-3], wherein the concentration of the trivalent phosphorus compound is 100 mM to 200 mM in the oxidizing solution. [4-1] The method according to any one of [1] to [3-4], wherein the iodine is contained in the oxidation solution at a concentration of 150 mM to 550 mM. [4-2] The method according to [4-1], wherein the iodine is contained in the oxidation solution at a concentration of 150 mM to 350 mM. [4-3] The method according to [4-2], wherein the iodine is contained in the oxidation solution at a concentration of 150 mM to 300 mM. [4-4] The method according to [4-3], wherein the iodine is contained in the oxidation solution at a concentration of 150 mM to 250 mM. [5-1] The method according to any one of [1] to [4-4], wherein the molar ratio of iodine to trivalent phosphorus compound in the oxidation solution is 1.50:1.00 to 1.10:1.00. [5-2] The method according to [5-1], wherein the molar ratio of iodine to the trivalent phosphorus compound in the oxidation solution is 1.50:1.00 to 1.17 to 1.00. [5-3] The method according to [5-2], wherein the molar ratio of iodine to the trivalent phosphorus compound in the oxidation solution is 1.50:1.00 to 1.20 to 1.00. [5-4] The method according to [5-3], wherein the molar ratio of iodine to the trivalent phosphorus compound in the oxidation solution is 1.50:1.00 to 1.25:1.0.

[0019] <Manufacturing method characterized by a solvent> [6] The method according to any one of [1] to [5-4], wherein the organic solvent in the oxidation solution is pyridine. [7] The method according to [6], wherein the oxidation solution further contains acetonitrile, N,N-dimethylformamide, toluene, tetrahydrofuran, isopropyl alcohol, and / or dichloromethane. [8] The method according to [7], wherein the solvent of the oxidation solution is pyridine:water:acetonitrile, N,N-dimethylformamide, toluene, tetrahydrofuran, isopropyl alcohol or dichloromethane in a volume ratio of 1 to 90:1 to 50:0 to 90.

[0020] <Oxidation solution> [9] An oxidation solution for the production of a mixed P=O / P=S backbone oligonucleic acid, comprising: a) iodine; b) 100 mM to 500 mM, preferably 100 mM to 300 mM, more preferably 100 mM to 250 mM, still more preferably 100 mM to 200 mM of a compound of the formula:

[0021] [wherein, R １ , R ２ and R ３ may be the same or different and independently represent a C ６－１４ aryl group or a C １－６ alkyl group, and two Rs may be bonded to each other and bonded to other adjacent atoms to form a ring], a trivalent phosphorus compound represented by; c) an organic solvent, preferably pyridine or a C １－６ alkyl-substituted pyridine, and d) water.

[10] a) 150 mM to 550 mM of iodine; b) 100 mM to 500 mM of the trivalent phosphorus compound; c) pyridine or a C １－６An oxidation solution according to [9], comprising an alkyl-substituted pyridine and d) water, wherein the molar ratio of iodine to the trivalent phosphorus compound is 1.50:1.00 to 1.10:1.00.

[11] An oxidation solution according to [9] or

[10] , wherein c) is pyridine.

[12] An oxidation solution according to any one of [9] to

[11] , wherein the oxidation solution further comprises acetonitrile, N,N-dimethylformamide, toluene, tetrahydrofuran, isopropyl alcohol, and / or dichloromethane.

[0022] The trivalent phosphorus compounds, such as triphenylphosphine, contained in the oxidation solution of the present invention are relatively stable to air and light, are non-hygroscopic, and minimize variations in additive concentration from one preparation to the next. As a result, the solution exhibits a PS-PO derivative suppression effect equivalent to or better than conventional commercially available products and prior art.

[0023] <Manufacturing method characterized by trivalent phosphorus compounds> The present invention, in one embodiment, is a method for producing mixed P=O / P=S backbone oligonucleotides, comprising iodine, an organic solvent, preferably pyridine or C １－６ Using an oxidation solution containing alkyl-substituted pyridine and water, the formula:

[0024] [In the formula, R １１ C may be replaced with CN. １－６ The process includes oxidizing the phosphite triester moiety of formula (I), represented by [formula (I)], to the phosphodiester moiety of formula (II), wherein the oxidizing solution further comprises:

[0025] [In the formula, R １ , R ２ and R ３ C may be the same or different, and may be substituted independently. ６－１４ Aryl group or C １－６The present invention relates to the method, characterized in that a trivalent phosphorus compound represented by [an alkyl group, in which two Rs are bonded to each other and may bond to other adjacent atoms to form a ring] is contained in an oxidizing solution at a concentration of 100 mM to 500 mM, preferably 100 mM to 300 mM, more preferably 100 mM to 250 mM, and even more preferably 100 mM to 200 mM.

[0026] In the method of the present invention, "mixed P=O / P=S backbone oligonucleotide" means an oligonucleotide containing a mixture of phosphodiester (P=O) and phosphorothioate (P=S). Here, "oligonucleotide" typically means a sequence of nucleotides with a length of 10 to 40 base pairs, preferably 10 to 25 base pairs, and may also be short nucleotides of about 20 base pairs or less. Oligonucleotides are also called "nucleic acid oligomers" or "oligonucleotides".

[0027] In one embodiment, the present invention relates to a method in which the oxidizing solution selectively oxidizes the phosphite triester portion of formula (I) to the phosphodiester portion of formula (II) without oxidizing the phosphorothioate nucleotide bonds in the mixed P=O / P=S backbone oligonucleotide.

[0028] In equations (I) and (II), R １１ "C which may be replaced by CN" １－６ "Alkyl group" refers to a linear or branched hydrocarbon group having 1 to 6 carbon atoms, which may be substituted with a cyano group: CN, and is preferably cyanoethyl.

[0029] In formulas (I) and (II), the nucleic acid base that may be protected by the protecting group represented by B is not particularly limited. Examples of such nucleic acid bases include adenine, cytosine, guanine, uracil, thymine, 5-methylcytosine, pseudouracil, and 1-methylpseudracil. Furthermore, the nucleic acid base may be substituted with substituents. Examples of such substituents include halogen atoms such as fluoro, chloro, bromo, and iodo groups; acyl groups such as acetyl groups; alkyl groups such as methyl and elyx groups; arylalkyl groups such as benzyl groups; alkoxy groups such as methoxy groups; alkoxyalkyl groups such as methoxyethyl groups; cyanoalkyl groups such as cyanoethyl groups; hydroxyl groups; hydroxyalkyl groups; acyloxymethyl groups; amino groups; monoalkylamino groups; dialkylamino groups; carboxyl groups; cyano groups; and nitro groups, as well as combinations of two or more substituents thereof.

[0030] In formulas (I) and (II), when R represents a protected hydroxyl group, the protecting group can be any that can be used in the amidite process, for example, 2'-tert-butyldimethylsilyl (TBDMS) group, 2'-bis(2-acetoxy)methyl (ACE) group, 2'-(triisopropylsilyloxy)methyl (TOM) group, 2'-(2-cyanoethoxy)ethyl (CEE) group, 2'-(2-cyanoethoxy)methyl (CEM) group, 2'-p-toluylsulfonylethoxymethyl (TEM) group, and 2'-EMM group (International Publication 2006 / 022323), as well as those described in International Publications 2013 / 027843 and 2019 / 208571.

[0031] The "oxidizing solution" used in the method of the present invention is used in the step of oxidizing the generated phosphite ester in the solid-phase synthesis of oligonucleotides, and typically contains iodine, an organic solvent, and water. In this embodiment, the organic solvent is preferably pyridine or C １－６ It is an alkyl-substituted pyridine.

[0032] The "oxidizing solution" used in the method of the present invention is further defined by formula:

[0033] [In the formula, R １ , R ２ and R ３ C may be the same or different, and may be substituted independently. ６－１４ Aryl group or C １－６ The product contains a trivalent phosphorus compound represented by [an alkyl group, where two Rs are bonded to each other and may bond to other adjacent atoms to form a ring] at a concentration of 100 mM to 500 mM, preferably 100 mM to 300 mM, more preferably 100 mM to 250 mM, and even more preferably 100 mM to 200 mM.

[0034] Here, "C ６－１４ The term "aryl group" refers to an aromatic hydrocarbon group having 6 to 14 carbon atoms, and includes, but is not limited to, phenyl, 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, and 9-anthryl. ６－１４ The "aryl group" is C １－６ Alkyl alkyl group, C １－６ Alkoxy groups and C ３－１０ The group may be substituted with one to three substituents selected from cycloalkyl groups. Examples of substituted aryl groups include, but are not limited to, toluyl and methoxyphenyl.

[0035] Here, "C １－６ The term "alkoxy group" refers to an (linear or branched hydrocarbon group having 1-6 carbon atoms) -O- group, and includes, but is not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, and hexyloxy. ３－１０ "Cycloalkyl group" means a cyclic hydrocarbon group having 3 to 10 carbon atoms, and includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.2.1]octyl, and adamantyl.

[0036] "C １－６"Alkyl group" means a linear or branched hydrocarbon group having 1 to 6 carbon atoms, and includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl.

[0037] In the present invention, R １ , R ２ and R ３ Among these, an example of a structure in which two or more R atoms bond to each other and then bond to other adjacent atoms to form a ring is the following structural formula:

[0038] Examples of compounds shown are, but are not limited to, those indicated by [the symbol].

[0039] The trivalent phosphorus compound used in the method of the present invention is preferably of formula:

[0040] [In the formula, R １ , R ２ and R ３ The compound is represented by [which has the same meaning as above], and more preferably, in such formula, R １ , R ２ and R ３ However, C may be substituted independently. ６－１４ Aryl group, preferably unsubstituted C ６－１４ Aryl group, more preferably R １ , R ２ and R ３ All are identical non-substituted C ６－１４ It is an aryl group. In light of the purpose of mass-producing oligonucleotides, from the viewpoint of chemical stability, safety, and odor, preferably, in the above formula, R １ , R ２ and R ３This is triphenylphosphine, where all elements are phenyl. Triphenylphosphine is relatively stable to air and light, is non-hygroscopic, and minimizes variations in additive concentration between preparations. Specifically, by including triphenylphosphine in the oxidation solution, it has been confirmed that it has an effect of suppressing the formation of PS-PO variants that is equivalent to or better than that of commercially available products and prior art, which unexpectedly produce PS-PO variants as by-products when dealing with the desired mixed P=O / P=S backbone oligonucleotides.

[0041] It has been found that the concentration of the trivalent phosphorus compound used in the oxidation solution is preferably above a certain concentration. Therefore, in the present invention, the trivalent phosphorus compound is contained in the oxidation solution at a concentration of 100 mM to 500 mM, preferably 100 mM to 300 mM, more preferably 100 mM to 250 mM, and even more preferably 100 mM to 200 mM.

[0042] In the method of the present invention, in a preferred embodiment, iodine is contained in the oxidation solution at a concentration of 150 mM to 550 mM, preferably 150 mM to 350 mM, more preferably 150 mM to 300 mM, and even more preferably 150 mM to 250 mM.

[0043] In the method of the present invention, in another preferred embodiment, the molar ratio of iodine to trivalent phosphorus compound in the oxidation solution is 1.50:1.00 to 1.10:1.00, preferably 1.50:1.00 to 1.17:1.0, more preferably 1.50:1.00 to 1.20:1.00, and even more preferably 1.50:1.00 to 1.25:1.00.

[0044] <Manufacturing method characterized by the solvent> In a preferred embodiment, the present invention relates to a method of the present invention in which the oxidizing solution contains pyridine. Preferably, the oxidizing solution further comprises acetonitrile (MeCN), N,N-dimethylformamide (DMF), toluene, tetrahydrofuran (THF), isopropyl alcohol (IPA), and / or dichloromethane (DCM).

[0045] The present invention relates, in another embodiment, to a method for which the solvent of the oxidation solution is pyridine:water:acetonitrile, N,N-dimethylformamide, toluene, tetrahydrofuran, isopropyl alcohol, or dichloromethane in a volume ratio of 1 to 90:1 to 50:0 to 90. Preferably, the ratio is 10 to 90:5 to 25:0 to 60, and more preferably 20 to 90:5 to 15:0 to 50.

[0046] Here, we will compare the method for producing mixed P=O / P=S backbone oligonucleotides of the present invention, which is characterized by the addition of a trivalent phosphorus compound to an oxidation solution, with other patent documents. In one embodiment of the method of the present invention, an oxidation solution containing iodine and triphenylphosphine, an organic solvent, and water as the solvent is used. This oxidation solution does not require maturation and can be used immediately after preparation. In WO2020-196890 (Patent Document 4), a phosphorus-based or sulfur-based antioxidant is used to reduce the by-product formation of PS-PO variants, but the step in which it is used is a phosphoramidite coupling step, not a step in oxidizing the phosphite ester in the present invention. Thus, none of the above patent documents describe or suggest the use of a specific concentration of a trivalent phosphorus compound in the oxidation step.

[0047] <Oxidizing Solution> In another embodiment, the present invention provides an oxidizing solution for the production of mixed P=O / P=S backbone oligonucleotides, comprising: a) iodine, b) 100 mM to 500 mM, preferably 100 mM to 300 mM, more preferably 100 mM to 250 mM, and even more preferably 100 mM to 200 mM, of formula:

[0048] [In the formula, R １ , R ２ and R ３ C may be the same or different, and may be substituted independently. ６－１４ Aryl group or C １－６ A trivalent phosphorus compound represented by [an alkyl group, where two Rs are bonded to each other and may bond to other adjacent atoms to form a ring], c) an organic solvent, preferably pyridine or C １－６This relates to an oxidation solution containing alkyl-substituted pyridine and d) water. The definitions of the symbols in the formula and the various terms are the same as above.

[0049] Preferably, the oxidizing solution of the present invention comprises: a) 150 mM to 550 mM iodine, b) 100 mM to 500 mM of the trivalent phosphorus compound, and c) pyridine or C １－６ The mixture comprises an alkyl-substituted pyridine and d) water, and more preferably, the molar ratio of iodine to the trivalent phosphorus compound is 1.50:1.00 to 1.10:1.00. The preferred organic solvent is pyridine. More preferably, the mixture further comprises acetonitrile, N,N-dimethylformamide, toluene, tetrahydrofuran, isopropyl alcohol, and / or dichloromethane.

[0050] The present invention will be described in detail below with reference to manufacturing examples and embodiments, but it should be noted that these are merely illustrative examples and do not limit the scope of the present invention.

[0051] Manufacturing Example 1 Synthesis of 20mer DNA represented by Formula 1: 5'-C(D)^T(D)*G(D)*C(D)*T(D)^A(D)^G(D)^C(D)^C(D)^T(D)^C(D)^T(D)^G(D)^G(D)^A(D)^T(D)*T(D)*T(D)*G(D)^A(D)-3' [wherein "^" indicates a phosphorothioate bond and "*" indicates a phosphodiester bond] Using an AKTA Oligopilot 100 as the nucleic acid synthesizer and a NittoPhase HL Unilinker 350 as the solid support, oligonucleotides consisting of the sequence of Formula 1 were synthesized on a scale of 250 nmol from the 3' side to the 5' side by solid-phase synthesis using the phosphoramidite method. The reagents used for solid-phase synthesis are shown in Table 1. After solid-phase synthesis, ammonium hydroxide was used to cleave the oligonucleotide from the solid support and deprotect the base portion to obtain crude oligonucleotides.

[0052] Table 1 Here, the deblocking solution is a deprotection solution. The oxidizing agent used was either a newly prepared solution or a commercially available product.

[0053] Reference Examples 1 and 2 and Examples 1 and 2 Preparation of a mixture containing iodine / triphenylphosphine in a pyridine / water = 9 / 1 solution Triphenylphosphine (PPh ３ Triphenylphosphine was added to a solution of water and pyridine at room temperature using the respective doses listed in Table 2 for iodine, water, and pyridine. After confirming the dissolution of triphenylphosphine, iodine was added, and the mixture was stirred for 1 hour before use. Table 2

[0054] Example 3 Preparation of a mixture containing 150 mM iodine / 100 mM triphenylphosphine in a pyridine / N,N-dimethylformamide:water = 6 / 3 / 1 solution 7.87 g of triphenylphosphine was added to a solution of 30 ml of water, 90 ml of N,N-dimethylformamide, and 180 ml of pyridine at room temperature. After confirming the dissolution of triphenylphosphine, 11.42 g of iodine was added, and the mixture was stirred for 1 hour before use.

[0055] Example 4 Preparation of a mixture containing 150 mM iodine and 100 mM triphenylphosphine in a pyridine / acetonitrile / water = 6 / 3 / 1 solution. 7.87 g of triphenylphosphine was added to a solution of 30 ml of water, 90 ml of acetonitrile, and 180 ml of pyridine at room temperature. After confirming the dissolution of triphenylphosphine, 11.42 g of iodine was added, and the mixture was stirred for 1 hour before use.

[0056] The following comparative mixtures were prepared: Comparative Example 1: A mixture containing commercially available 50 mM iodine in a pyridine / water = 9 / 1 solution. Comparative Example 2: A mixture containing newly prepared 50 mM iodine in a pyridine / water = 9 / 1 solution.

[0057] Comparative Example 3: A mixture containing newly prepared 50 mM iodine and 50 mM potassium iodide in a pyridine / water = 9 / 1 solution: 2.49 g of the potassium iodide mixture described in Patent Document 3 was added to a solution of 30 ml of water and 270 ml of pyridine at room temperature. After confirming the dissolution of potassium iodide, 3.81 g of iodine was added, and the mixture was stirred for 1 hour before use. Comparative Example 4: A mixture containing newly prepared 50 mM iodine and 100 mM potassium iodide in a pyridine / water = 9 / 1 solution: 4.98 g of the potassium iodide mixture described in Patent Document 3 was added to a solution of 30 ml of water and 270 ml of pyridine at room temperature. After confirming the dissolution of potassium iodide, 3.81 g of iodine was added, and the mixture was stirred for 1 hour before use.

[0058] Measurement Method 1: Measurement of Purity and PS-PO Conjugate Content (%) (1) The purity and PS-PO convertible content (%) were measured for the mixtures prepared in Reference Examples 1 and 2, Examples 1-4, and Comparative Examples 1-4. In detail, the purity of the crude oligonucleotide in each mixture was measured by ultra-high performance liquid chromatography (UHPLC) under the conditions in Table 3 and calculated using the formula: Peak area of ​​target oligonucleotide / Total peak area (%). The PS-PO convertible content (%) was calculated by mass spectrometry using the formula: Ionic intensity of PS-PO convertible / Ionic intensity of target oligonucleotide (%). Here, the PS-PO convertible content (%) refers to the percentage ratio of molecules in which at least one PS bond has been converted to a PO bond, relative to the total number of molecules. Table 3

[0059] The results obtained are shown in Table 4. Table 4

[0060] The results in Table 4 show the following: The composition of the oxidizing agent in Reference Example 1 resulted in a higher PS-PO derivative content compared to the commercially available oxidizing agent shown in Comparative Example 1 and the oxidizing agent from the prior art (Non-Patent Literature 3) shown in Comparative Example 3. In Reference Example 2, when the amount of triphenylphosphine added to the oxidizing agent was increased compared to Reference Example 1, the formation of the PS-PO derivative was suppressed more effectively than in Reference Example 1, and this suppression was at a level equivalent to that of the oxidizing agent from the prior art (Non-Patent Literature 3) shown in Comparative Example 4.

[0061] In Reference Example 2, iodine and triphenylphosphine were used as oxidizing agents, and it is assumed that the same amount of iodide ions as in Comparative Example 4 were generated in the solution. This supports the reason why the suppression of PS-PO derivative formation was equivalent. On the other hand, the results of Reference Examples 1 and 2 show that further suppression of PS-PO derivative formation can be expected by increasing the amount of triphenylphosphine added. From Comparative Examples 1 and 2, it can be concluded that a similar effect can be expected by increasing the amount of potassium iodide added. However, at high concentrations of potassium iodide, inorganic salts precipitate, which raises concerns about pipe blockage during solid-phase synthesis. On the other hand, the triphenylphosphine and iodide ion products used in Examples 1 and 2 are easily soluble in organic solvents, so the possibility of precipitation is low.

[0062] In Examples 1 and 2, it was confirmed that the formation of PS-PO variants could be suppressed at a lower level than that of prior art oxidizing agents by adding 100 mM or more of triphenylphosphine. Regarding the target nucleic acid purity, it was equivalent to that of commercially available oxidizing agents and prior art oxidizing agents, indicating that no decrease in nucleic acid purity was observed with the addition of triphenylphosphine.

[0063] Measurement Method 2: Measurement of Purity and PS-PO Conjugate Content (%) (2) The purity and PS-PO convertible content (%) of the mixtures of Reference Examples 3 and 4 and Examples 5-8, which were prepared separately from the above examples, were measured. The results obtained are shown in Table 5. Table 5

[0064] The results in Table 5 show the following: A decrease in the PS-PO variant content was observed as the amount of triphenylphosphine added increased. On the other hand, in Examples 7 and 8, a decrease in the target nucleic acid purity was observed by increasing the amount of triphenylphosphine added.

[0065] Measurement Method 3: Measurement of Purity and PS-PO Conversion Content (%) of Mixtures Containing Various Trivalent Phosphorus Compounds The purity and PS-PO conversion content (%) of the mixtures of Examples 9-11, prepared using the following trivalent phosphorus compounds instead of triphenylphosphine and in substantially the same manner as in the above examples, were measured. The results are shown in Table 6. Table 6

[0066] The results in Table 6 show the following: Similar inhibitory effects on the formation of PS-PO derivatives were confirmed for trivalent phosphorus compounds other than triphenylphosphine.

[0067] Measurement Method 4: Measurement of Purity and PS-PO Conversion Content (%) of Mixtures Further Containing Various Solvents The purity and PS-PO conversion content (%) of the mixtures of Examples 12-17, prepared by substantially the same method as in the above examples, were measured, with the following solvents further contained instead of acetonitrile. The results are shown in Table 7. Table 7

[0068] The results in Table 7 show the following: While a mixture of pyridine and water is generally used as the solvent for the oxidizing agent in the solid-phase synthesis of oligonucleotides, it was confirmed that the inhibitory effect on the formation of PS-PO variants is maintained even when acetonitrile, N,N-dimethylformamide, toluene, tetrahydrofuran, isopropyl alcohol, and / or dichloromethane are added as needed.

[0069] The oxidizing agent according to the present invention can significantly reduce the amount of PS-PO variants produced as by-products in the process of manufacturing oligonucleotides compared to commercially available or prior art oxidizing agents. Therefore, the present invention contributes to the production of high-quality oligonucleotides.

Claims

1. A method for producing a mixed P=O / P=S backbone oligonucleotide, comprising an oxidation solution containing iodine, an organic solvent, and water, wherein the formula is: [In the formula, R １１ C may be replaced with CN. １－６ The process includes oxidizing the phosphite triester moiety of formula (I), represented by [formula (I)], to the phosphodiester moiety of formula (II), wherein the oxidizing solution further comprises: [In the formula, R １ , R ２ and R ３ C may be the same or different, and may be substituted independently. ６－１４ Aryl group or C １－６ The method is characterized by containing a trivalent phosphorus compound represented by [an alkyl group, in which two Rs are bonded to each other and may bond to other adjacent atoms to form a ring] in an oxidation solution at a concentration of 100 mM to 500 mM.

2. The method according to claim 1, wherein the oxidizing solution selectively oxidizes the phosphite triester portion of formula (I) to the phosphodiester portion of formula (II) without oxidizing the phosphorothioate nucleotide bonds in the mixed P=O / P=S backbone oligonucleotide.

3. The method according to claim 1, wherein the concentration of the trivalent phosphorus compound in the oxidizing solution is 100 mM to 500 mM.

4. The method according to claim 1, wherein the iodine is contained in the oxidation solution at a concentration of 150 mM to 550 mM.

5. The method according to claim 1, wherein the molar ratio of iodine to trivalent phosphorus compound in the oxidation solution is 1.50:1.00 to 1.10:1.

00.

6. The method according to claim 1, wherein the organic solvent in the oxidation solution is pyridine.

7. The method according to claim 6, wherein the oxidizing solution further comprises acetonitrile, N,N-dimethylformamide, toluene, tetrahydrofuran, isopropyl alcohol, and / or dichloromethane.

8. The method according to claim 7, wherein the solvent of the oxidation solution has a volume ratio of pyridine:water:acetonitrile, N,N-dimethylformamide, toluene, tetrahydrofuran, isopropyl alcohol, or dichloromethane of 1 to 90:1 to 50:0 to 90.

9. An oxidation solution for the production of a mixed P=O / P=S backbone oligonucleic acid, comprising: a) iodine, b) 100 mM to 500 mM of a trivalent phosphorus compound represented by the formula: [wherein, R １ , R ２ and R ３ may be the same or different and are independently optionally substituted C ６－１４ aryl group or C １－６ alkyl group, and two Rs may be bonded to each other and bonded to other adjacent atoms to form a ring], c) an organic solvent, and d) water.

10. a) 150 mM to 550 mM iodine, b) 100 mM to 500 mM of the aforementioned trivalent phosphorus compound, c) pyridine or C １－６ The oxidation solution according to claim 9, comprising an alkyl-substituted pyridine and d) water, wherein the molar ratio of iodine to the trivalent phosphorus compound is 1.50:1.00 to 1.10:1.

00.

11. The oxidizing solution according to claim 9, wherein c) is pyridine.

12. The oxidation solution according to claim 9, wherein the oxidation solution further comprises acetonitrile, N,N-dimethylformamide, toluene, tetrahydrofuran, isopropyl alcohol, and / or dichloromethane.

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