Method for producing oligonucleotides having phosphorothioate site

Abstract

To provide a method for producing oligonucleotides having a phosphorothioate site.SOLUTION: Provided is a method for producing oligonucleotide having a phosphorothioate site, including a step of obtaining a phosphite or phosphite diester by condensing a nucleoside, nucleotide, or oligonucleotide in which the 5'-position hydroxyl group is not protected and other groups may be protected with a protecting group used in nucleic acid synthesis or may be bound to a solid-phase support, and a nucleoside, nucleotide, or oligonucleotide in which the 3'-position hydroxyl group or 3'-position amino group has been modified by a method for forming a phosphite or phosphite diester and other groups may be protected with a protecting group used in nucleic acid synthesis, in the presence of an antioxidant.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing an oligonucleotide having a phosphorothioate moiety, and is useful in the field of nucleic acid synthesis. [Background technology]

[0002] Nucleic acid drugs such as antisense, siRNA (small interfering RNA), and aptamers are attracting attention as the next generation of drugs following antibody drugs. Nucleic acid drugs are expected to have high specificity and efficacy like antibody drugs, but they can be produced by chemical synthesis like conventional drugs consisting of small molecular weight compounds. In research and development of new nucleic acid drugs, modified nucleic acids in which the phosphorus atom at the binding site between nucleic acids has been modified have been examined. Representative types of nucleic acid drugs have phosphorothioate sites (phosphorothioate internucleic acid binding sites) where the phosphorus atom is S-substituted, and examples of these include Fomivirsen, Mipomersen, Oblimersen, and Alicaforsen. Methods for producing oligonucleotides having phosphorothioate sites include solid-phase synthesis (solid-phase method) and liquid-phase method. In order to eliminate the drawbacks of the solid-phase method and the liquid-phase method, a liquid-phase method using a pseudo-solid-phase protecting group has also been developed (Patent Documents 1 to 9). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2012 / 157723 [Patent Document 2] International Publication No. 2013 / 122236 [Patent Document 3] International Publication No. 2017 / 104836 [Patent Document 4] International Publication No. 2005 / 070859 [Patent Document 5] International Publication No. 2013 / 179412 [Patent Document 6] International Publication No. 2014 / 077292 [Patent Document 7] International Publication No. 2017 / 086397 [Patent Document 8] International Publication No. 2018 / 203574 [Patent Document 9] International Publication No. 2018 / 212236 Summary of the Invention [Problem to be solved by the invention]

[0004] In the synthesis of oligonucleotides having phosphorothioate sites, the phosphoramidite method and the H-phosphonate method are currently widely used, and the dihalophosphine method and the oxazaphospholidine method are also known. In the phosphoramidite method, the dihalophosphine method and the oxazaphospholidine method, a phosphite is synthesized, and in the H-phosphonate method, a phosphite diester is synthesized, and each is subjected to a sulfurization reaction to obtain the target oligonucleotide having phosphorothioate sites. However, the inventor has newly found, as a result of his investigation, that when synthesis is performed according to such a method, at the stage where the phosphite or phosphite diester is produced, an oxidation reaction of the phosphorus atom also proceeds, and a phosphotriester or phosphodiester (hereinafter sometimes referred to as "PO impurity") is by-produced, which inhibits the subsequent sulfurization reaction, and the target phosphorothioate oligonucleotide cannot be efficiently synthesized. This "PO impurity" is an impurity in which the original phosphorothioate site is oxidized to a phosphotriester or phosphodiester. An object of the present invention is to solve this newly discovered problem and to provide a method for producing an oligonucleotide having a desired phosphorothioate site in higher yield. [Means for solving the problem]

[0005] The present inventors have found that this new problem can be solved by making a specific antioxidant coexist in the step of synthesizing a phosphite using a method for forming a phosphite (e.g., the phosphoramidite method, the dihalophosphine method, and the oxazaphospholidine method), or in the step of synthesizing a phosphite diester using a method for forming a phosphite (e.g., the H-phosphonate method), and have thus completed the present invention. The present invention is as follows.

[0006] [1] A method for producing an oligonucleotide having a phosphorothioate site, comprising the step of condensing a nucleoside, nucleotide or oligonucleotide (hereinafter sometimes referred to as "nucleic acid A") in which the 5'-position hydroxyl group is not protected and other groups may be protected with a protecting group used in nucleic acid synthesis or may be bound to a solid phase support, with a nucleoside, nucleotide or oligonucleotide (hereinafter sometimes referred to as "nucleic acid B") in which the 3'-position hydroxyl group or 3'-position amino group has been modified by a method for forming a phosphite or phosphite diester and other groups may be protected with a protecting group used in nucleic acid synthesis, in the presence of an antioxidant to obtain a phosphite or phosphite diester (step (1)). [2] A method for producing an oligonucleotide having a phosphorothioate site described in [1] above, in which the 3'-position hydroxyl group or the 3'-position amino group in nucleic acid B is modified by a method selected from the phosphoramidite method, the H-phosphonate method, the dihalophosphine method and the oxazaphosphoridine method. [3] A method for producing an oligonucleotide having a phosphorothioate site described in [1] or [2] above, further comprising a step (step (2)) of reacting the phosphite or phosphite diester obtained in step (1) with a sulfurizing agent. [4] (1) a step of condensing, in the presence of an antioxidant, a nucleoside, nucleotide, or oligonucleotide (hereinafter sometimes referred to as "nucleic acid A") in which the 5'-position hydroxyl group is not protected and other groups may be protected with a protecting group used in nucleic acid synthesis or may be bound to a solid phase support, with a nucleoside, nucleotide, or oligonucleotide (hereinafter sometimes referred to as "nucleic acid B") in which the 3'-position hydroxyl group or the 3'-position amino group has been modified by a method selected from the phosphoramidite method, the H-phosphonate method, the dihalophosphine method, and the oxazaphospholidine method and other groups may be protected with a protecting group used in nucleic acid synthesis, to obtain a phosphite or a phosphite diester; (2) A method for producing an oligonucleotide having a phosphorothioate site, comprising the step of reacting the obtained phosphite or phosphite diester with a sulfurizing agent.

[0007] [5] A method for producing an oligonucleotide having a phosphorothioate site according to any one of [1] to [4] above, wherein the antioxidant is selected from phosphorus-based antioxidants, sulfur-based antioxidants, and phosphorus-sulfur-based antioxidants.

[0008] [6] Antioxidants: (1) P-(R)3 (2) P-(OR)3 (3) P(R)2(OR) (4)PR(OR)2 (5) PH(O)(R)2 (6) PH(O)(OR)2, and (7) PH(O)R(OR) (In the above formulas (1) to (7), R may be the same or different and independently represent an optionally substituted aryl group or a lower alkyl group, and two R may be bonded to each other and form a ring together with another adjacent atom; Here, the antioxidant may be a dimer in which the R moieties of the same or different antioxidants are bonded together. A phosphorus-based antioxidant selected from (8) RSR, and (9)

[0009] [ka]

[0010] (In the above formulas (8) and (9), R may be the same or different and each independently represents a lower alkyl group or an optionally substituted aryl group; Here, the antioxidant may be a dimer in which the R moieties of the same or different antioxidants are bonded together. A sulfur-based antioxidant selected from the group consisting of: (10)(OR)2P(S)-SMSP(S)(OR)2 (In the formula, R may be the same or different and independently represents a lower alkyl group or an optionally substituted aryl group, and M represents Zn, Ni or Cu; Here, the antioxidant may be a dimer in which the R moieties of the same or different antioxidants are bonded together. and a phosphorus-sulfur antioxidant represented by the formula (1): [7] Antioxidants: (1) P-(R)3 (2) P-(OR)3 (3) P(R)2(OR) (4) PR(OR)2, and (5) PH(O)R(OR) (In the above formulas (1) to (5), R may be the same or different and independently represent an optionally substituted aryl group or a lower alkyl group, and two R may be bonded to each other and form a ring together with another adjacent atom; Here, the antioxidant may be a dimer in which the R moieties of the same or different antioxidants are bonded together. A phosphorus-based antioxidant selected from the group consisting of (6)

[0011] [ka]

[0012] (In the above formula (6), R may be the same or different and each independently represents a lower alkyl group or an optionally substituted aryl group; Here, the antioxidant may be a dimer in which the R moieties of the same or different antioxidants are bonded together. and a sulfur-based antioxidant represented by the formula (1): [8] The method for producing an oligonucleotide having a phosphorothioate site according to any one of the above [1] to [4], wherein the antioxidant is selected from the group consisting of triphenylphosphine, methyldiphenylphosphine, triethyl phosphite, ethoxydiphenylphosphine, diethoxyphenylphosphine, 9,10-dihydro-9-oxa-10-phosphaphenanthrene 10-oxide (phosphorus-based antioxidants); and isobutylene sulfide (sulfur-based antioxidants). [9] The method for producing an oligonucleotide having a phosphorothioate site according to any one of the above [1] to [4], wherein the antioxidant is a phosphorus-based antioxidant.

[10] The method for producing an oligonucleotide having a phosphorothioate site according to any one of the above [1] to [5], wherein the antioxidant is a phosphine represented by P-(R)3.

[0013]

[11] A method for producing an oligonucleotide having a phosphorothioate site according to any one of [1] to

[10] above, wherein nucleic acid A is a nucleoside, nucleotide or oligonucleotide in which the 5'-hydroxyl group is not protected and other groups may be protected with a protecting group used in nucleic acid synthesis.

[0014]

[12] Nucleic acid A is a nucleoside, nucleotide or oligonucleotide (hereinafter sometimes referred to as "nucleic acid a") in which the 5'-position hydroxyl group is not protected, and at least one group selected from the amino group and imino group of the nucleic acid base, the 2'-position hydroxyl group, the 3'-position hydroxyl group and the 3'-position amino group of the ribose residue, and the 3'-position hydroxyl group and the 3'-position amino group of the deoxyribose residue is protected with a protecting group that is not removable under acidic conditions but is removable under basic conditions (hereinafter sometimes referred to as "pseudo solid-phase protecting group"), and other groups may be further protected with a protecting group used in nucleic acid synthesis (preferably, the 3'-position of the ribose residue and the deoxyribose residue is a hydroxyl group), or A nucleoside, nucleotide or oligonucleotide (hereinafter sometimes referred to as "nucleic acid α") in which the 5'-position hydroxyl group is not protected, one OH of the phosphate group at the 3'-terminus is replaced with -OLn1-OH (wherein Ln1 represents an organic group), the hydroxyl group of -OLn1-OH is protected with a protecting group that is not removable under acidic conditions but is removable under basic conditions, and other groups may be further protected with protecting groups used in nucleic acid synthesis, and nucleic acid B is a nucleoside, nucleotide or oligonucleotide in which the 3'-position hydroxyl group or the 3'-position amino group is protected with a protecting group that is not removable under acidic conditions but is removable under basic conditions, and the other groups may be further protected with protecting groups used in nucleic acid synthesis, a method for producing an oligonucleotide having a phosphorothioate site according to any one of the above [1] to

[11] , which is a nucleoside, nucleotide or oligonucleotide (hereinafter sometimes referred to as "nucleic acid b") which has been modified by a method selected from the group consisting of a phosphorothioate method, a dihalophosphine method and an oxazaphosphoridine method, and in which the 5'-hydroxyl group is protected by a temporary protecting group removable under acidic conditions, and in which other groups may be further protected by a protecting group which is not removed under acidic conditions but is removable under basic conditions (hereinafter sometimes referred to as a "pseudo solid-phase protecting group") and a protecting group used in nucleic acid synthesis.

[0015]

[13] A method for producing an oligonucleotide having a phosphorothioate site according to any one of the above [1] to

[12] , wherein nucleic acid B is a nucleoside, nucleotide or oligonucleotide in which the 3'-hydroxyl group has been modified by a method for forming a phosphite or a phosphorous diester (preferably a method selected from the group consisting of the phosphoramidite method, the H-phosphonate method, the dihalophosphine method and the oxazaphospholidine method) and other groups may be protected by a protecting group used in nucleic acid synthesis.

[0016]

[14] A method for producing an oligonucleotide having a phosphorothioate site according to any one of [3] to

[13] above, wherein the sulfurizing agent used in step (2) is selected from 5-[(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazole-3-thione (DDTT), 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent), 3H-1,2-benzodithiol-3-one, phenylacetyl disulfide (PADS), tetraethylthiuram disulfide (TETD), dipentamethylenethiuram tetrasulfide, 5-phenyl-3H-1,2,4-dithiazole-3-one (POS), 3-amino-1,2,4-dithiazole-5-thione (ADTT, xanthan hydride), N-[(2-cyanoethyl)thio]phthalimide and sulfur.

[0017]

[15] A method for producing an oligonucleotide having a phosphorothioate site according to any one of [1] to

[14] above, further comprising a step of removing all protecting groups from the obtained oligonucleotide having a phosphorothioate site and then isolating an unprotected oligonucleotide having a phosphorothioate site.

[0018]

[16] (1) A step of condensing, in the presence of an antioxidant, a nucleoside, nucleotide or oligonucleotide (nucleic acid A-1) (preferably, the 3'-position in nucleic acid A-1 is a hydroxyl group) in which the 3'-position hydroxyl group or the 3'-position amino group is not protected and other groups may be protected with a protecting group used in nucleic acid synthesis or may be bound to a solid-phase support, with a nucleoside, nucleotide or oligonucleotide (nucleic acid B-1) in which the 5'-position hydroxyl group has been modified by a method for forming a phosphite or phosphorous diester compound (preferably a method selected from the phosphoramidite method, the H-phosphonate method, the dihalophosphine method and the oxazaphospholidine method) and other groups may be protected with a protecting group used in nucleic acid synthesis, to obtain a phosphite or phosphorous diester compound (step (1-1)); A method for producing an oligonucleotide having a phosphorothioate site comprising:

[17] Further comprising a step (step (2-1)) of reacting the phosphite or phosphorous diester obtained in step (1-1) with a sulfurizing agent, A method for producing an oligonucleotide having a phosphorothioate site according to the above

[16] . Effect of the Invention

[0019] According to the present invention, in the stage of synthesizing a phosphite or phosphite diester using a method widely used in the art for forming a phosphite or phosphite diester, such as the phosphoramidite method or the H-phosphonate method, the by-production of a phosphotriester or phosphodiester ("PO impurity") can be suppressed by the coexistence of a specific antioxidant, and the subsequent sulfurization reaction can be carried out more efficiently. The "PO impurity" is an impurity in which a site that is originally phosphorothioated is oxidized and converted to a phosphodiester. Therefore, by carrying out the present invention in all condensation steps or in the condensation steps required in the nucleic acid chain extension in oligonucleotide synthesis, the oligonucleotide having the desired phosphorothioated site can be produced in a higher yield. In the production of an oligonucleotide having a phosphorothioated site, the nucleic acid extension cycle is appropriately repeated, so that even if a trace amount of impurity of 1% or less is produced in the reaction step for each base extension, the amount of impurity becomes large when the target oligonucleotide extension is produced. Therefore, in order to obtain high-purity oligonucleotides, it is essential to suppress the by-production of impurities in each individual reaction step, and the significance of the present invention, which suppresses the by-production of "PO impurities," is extremely great. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Unless otherwise specified in the context, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications and patents mentioned herein are incorporated by reference for the purpose of describing and disclosing, for example, the constructs and methodologies described in the publications that can be used in connection with the described invention.

[0021] [Method for producing oligonucleotides having phosphorothioate moieties] The production method of the present invention is a method for producing an oligonucleotide having a phosphorothioate site, which comprises the following step (1) and further comprises step (2):

[0022] Process (1) A step of condensing, in the presence of an antioxidant, a nucleoside, nucleotide or oligonucleotide (nucleic acid A) in which the 5'-position hydroxyl group is not protected and other groups may be protected with a protecting group used in nucleic acid synthesis or may be bound to a solid phase support, with a nucleoside, nucleotide or oligonucleotide (nucleic acid B) in which the 3'-position hydroxyl group or 3'-position amino group has been modified by a method for forming a phosphite or phosphorous diester compound (e.g., a method selected from the phosphoramidite method, the H-phosphonate method, the dihalophosphine method, and the oxazaphospholidine method) and other groups may be protected with a protecting group used in nucleic acid synthesis, to obtain a phosphite or phosphorous diester compound.

[0023] Process (2) A step of reacting the obtained phosphite or phosphorous diester with a sulfurizing agent.

[0024] In the process of extending a nucleotide chain by the phosphoramidite method known in the art, all or necessary internucleic acid bond sites can be phosphorothioated with high yield by carrying out the above-mentioned step (1) of the present invention and further step (2) in the extension step where a phosphorothioate site is to be introduced. The method for producing the oligonucleotide of the present invention can be combined with the method for producing an oligonucleotide in which the internucleic acid bond site is a phosphotriester bond or a phosphodiester bond that is commonly used in the field of nucleic acid synthesis (hereinafter sometimes referred to as "the method for producing a conventional oligonucleotide"), and the cycle of nucleic acid extension can be appropriately repeated according to the oligonucleotide having the desired phosphorothioate site, thereby extending the oligonucleotide having the phosphorothioate site to produce an oligonucleotide having the desired phosphorothioate site. Such an embodiment is also included in the scope of the present invention.

[0025] The above steps (1) and (2) of the present invention will be described in detail below.

[0026] The above steps (1) and (2) can be carried out under either solid-phase or liquid-phase reaction conditions, but are more preferably carried out under liquid-phase conditions.

[0027] Step (1) Condensation reaction in the presence of an antioxidant

[0028] The modification of the 3'-hydroxyl group or 3'-amino group of nucleic acid B in step (1) to form a phosphite or phosphorous diester derivative can be carried out by a method known in the art selected from, for example, the phosphoramidite method, the H-phosphonate method, the dihalophosphine method, and the oxazaphospholidine method. These methods are widely known to those skilled in the art, and the phosphite or phosphorous diester formed during the condensation reaction will also be readily understood by those skilled in the art. When a condensate is obtained by applying a method such as the phosphoramidite method, the H-phosphonate method, the dihalophosphine method, or the oxazaphosphoridine method using a nucleic acid B having an amino group at the 3' position instead of a nucleic acid B having a hydroxyl group at the 3' position, the bond between the 3' position and the phosphorus atom becomes an -NP bond instead of an -OP bond, but in this case as well, it will be referred to as a "phosphite" or "phosphorous diester" for convenience in this specification as in the case of an -OP bond. Similarly, the corresponding "PO impurity" will be referred to as a "phosphotriester" or "phosphodiester". Here, nucleic acid B is preferably a nucleoside, nucleotide or oligonucleotide in which the 3'-hydroxyl group has been modified by a method for forming a phosphite or a phosphorous diester and other groups may be protected with a protecting group used in nucleic acid synthesis.

[0029] Modification by the phosphoramidite method can be carried out with reference to, for example, SL Beaucage, Protocols for Oligonucleotides and Analogs (Chapter 3) 1993, pp. 33-62; MH Caruthers et al., Method in Enzymology 1987, pp. 154, 287-313; SL Beaucage and MH Caruthers, Tetrahedron Letters 1981, 22, 1859-1862; Japan Chemical Society, 5th Edition, Experimental Chemistry Lectures 16, Organic Compounds IV, pp. 377-381, 2010.

[0030] Modification using the H-phosphonate method can be carried out by referring to, for example, BF Froehler, Protocols for Oligonucleotides and Analogs (Chapter 4) 1993, pp. 63-80; Japan Chemical Society, 5th Edition, Experimental Chemistry Lectures 16, Organic Compounds IV, 2010, pp. 381-384, and the like.

[0031] Modification using dihalophosphine derivatives (dihalophosphine method) can be carried out by referring to, for example, Japan Chemical Society, 4th Edition, Experimental Chemistry Lectures 22, Metal Complexes and Transition Metal Clusters, 1999, pp. 426-431.

[0032] Modification using the oxazaphospholidine method can be carried out with reference to, for example, N. Oka et al., J. Am. Chem. Soc., 2008, 130, 16031-16037; N. Oka et al., Organic Letters., 2009, Vol. 11, No. 4, 967-970; International Publication No. 2011 / 08682, and the like.

[0033] Nucleic acid B, in which the 3'-position hydroxyl group or 3'-position amino group has been modified by a method for forming a phosphite or phosphorous diester compound (e.g., a method selected from the phosphoramidite method, the H-phosphonate method, the dihalophosphine method, and the oxazaphospholidine method), is condensed with nucleic acid A in the presence of an antioxidant to form the corresponding phosphite or phosphorous diester compound. Many improvements to the above-mentioned method for forming a phosphite or phosphorous diester are known in the art (see, for example, International Publication No. 2014 / 010250), and these can also be used in carrying out the present invention. In addition, when nucleic acid B modified by a method for forming a phosphite or phosphorous diester is commercially available, the commercially available product can also be used in carrying out the present invention.

[0034] The antioxidant used should preferably have sufficient antioxidant effect to prevent the by-production of phosphotriesters or phosphodiesters ("PO impurities") due to oxidation of phosphorus atoms in the phosphite or diphosphite produced, and should react with nucleic acid B (phosphoramidite, H-phosphonate, etc.) in the condensation step without adversely affecting the condensation step itself. Furthermore, when the step (2) following the condensation reaction is carried out under liquid phase conditions in the same reactor, it is preferable to select an antioxidant that does not adversely affect the subsequent reaction and does not induce the by-production of impurities. From this viewpoint, preferred antioxidants include, for example, phosphorus-based antioxidants, sulfur-based antioxidants, and phosphorus-sulfur-based antioxidants. Specific examples include antioxidants represented by the following formula:

[0035] Examples of phosphorus-based antioxidants include the following: (1) P-(R)3 (sometimes called "phosphines") (2) P-(OR)3 (sometimes called "phosphites") (3) P(R)2(OR) (sometimes called "phosphinites") (4) PR(OR)2 (sometimes called "phosphonite esters") (5) PH(O)(R)2 (sometimes called "phosphine oxides") (6) PH(O)(OR)2 (sometimes called "phosphonates") (7) PH(O)R(OR) (sometimes called "phosphinic acid esters") (In the above formulas (1) to (7), R may be the same or different and independently represent an optionally substituted aryl group or a lower alkyl group, and two R may be bonded to each other and form a ring together with another adjacent atom; Here, the antioxidant may be a dimer in which the R moieties of the same or different antioxidants are bonded together. The above dimer may, for example, be one represented by the following structural formula:

[0036] [ka]

[0037] In the present specification, the term "aryl group" refers to a group including "C 6-14 Aryl groups, for example, phenyl, 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, and 9-anthryl. The "aryl group" may be substituted with 1 to 3 substituents selected from a lower alkyl group, a lower alkoxy group, and a lower cycloalkyl group (eg, toluoyl, methoxyphenyl). In the present specification, the term "lower alkyl" includes "C 1-6 and "alkyl groups", such as 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. In the present specification, the term "lower alkoxy group" refers to a "C 1-6 Alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, and hexyloxy. In the present specification, the term "lower cycloalkyl group" includes "C 3-10 Cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.2.1]octyl, and adamantyl. The same applies to the other antioxidants described below.

[0038] Examples of sulfur-based antioxidants include the following: (8) RSR (9)

[0039] [ka]

[0040] (In the above formulas (8) and (9), R may be the same or different and each independently represents a lower alkyl group or an optionally substituted aryl group; Here, the antioxidant may be a dimer in which the R moieties of the same or different antioxidants are bonded together.

[0041] Examples of phosphorus-sulfur based antioxidants include the following: (10)(OR)2P(S)-SMSP(S)(OR)2 (In the formula, R may be the same or different and independently represents a lower alkyl group or an optionally substituted aryl group, and M represents Zn, Ni or Cu; Here, the antioxidant may be a dimer in which the R moieties of the same or different antioxidants are bonded together.

[0042] Among the above antioxidants, more preferred ones are phosphines represented by the formula P-(R)3, phosphorous esters (or phosphites) represented by the formula P-(OR)3, phosphinic acid esters represented by the formula P(R)2(OR), phosphonic acid esters represented by the formula PR(OR)2, phosphinic acid esters represented by the formula PH(O)R(OR),

[0043] [ka]

[0044] Examples of the sulfur-based antioxidant include those represented by the following formula:

[0045] As the phosphines represented by the formula P-(R)3, for example, triphenylphosphine and methyldiphenylphosphine are preferred. Among them, triphenylphosphine is more preferred. As the phosphite ester (or phosphite) represented by the formula P-(OR)3, for example, triethyl phosphite (P(OEt)3) is preferred. The phosphinites represented by the formula P(R)2(OR) are preferably, for example, ethoxydiphenylphosphine.The phosphonites represented by the formula PR(OR)2 are preferably, for example, diethoxyphenylphosphine.

[0046] Examples of the phosphinic acid esters represented by the formula: PH(O)R(OR) include:

[0047] [ka]

[0048] Preferred is 9,10-dihydro-9-oxa-10-phosphaphenanthrene 10-oxide represented by the following formula:

[0049] [ka]

[0050] As the sulfur-based antioxidant represented by the formula:

[0051] [ka]

[0052] Preferred is isobutylene sulfide represented by the following formula:

[0053] The amount of antioxidant used is preferably as small as possible in consideration of the effect on the subsequent reaction, and is usually preferably 0.1 to 2 mol, more preferably 0.2 to 1 mol, per mol of nucleic acid A used in the condensation reaction.

[0054] The solvent used when this step is carried out under liquid phase conditions is not particularly limited as long as it dissolves nucleic acid A, nucleic acid B, the antioxidant, etc. and does not adversely affect the reaction, and examples thereof include methylene chloride, acetonitrile, chloroform, tetrahydrofuran, acetone, ethyl acetate, CPME, toluene, cyclohexane, mixed solvents thereof, etc. Among these, chloroform, tetrahydrofuran, toluene, methylene chloride, acetonitrile, and mixed solvents thereof are preferred.

[0055] The concentrations of nucleic acid A and nucleic acid B in the solution used in this step are not particularly limited as long as they are dissolved in the solvent. The amount of nucleic acid B used is preferably 1 to 3 mol, more preferably 1.2 to 2.5 mol, per mol of nucleic acid A used.

[0056] The condensation reaction of this step can be carried out by adding an activator. For example, the activator of the phosphoramidite method includes tetrazole derivatives (e.g., 5-ethylthio-1H-tetrazole (ETT), 5-benzylthio-1H-tetrazole, etc.) and imidazole derivatives (e.g., 4,5-dicyanoimidazole, etc.) that are commonly used in the art. Preferably, it is a tetrazole derivative, more preferably 5-ethylthio-1H-tetrazole. The activator of the H-phosphonate method includes only a condensation agent, a combination of a condensation agent and a condensation additive, or pivaloyl chloride. Condensation agents alone include HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), HCTU (O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), Pybop (hexafluorophosphate (benzotriazol-1-yloxy) tripyrrolidinophosphonium), BopCl (bis(2-oxo-3-oxazolidinyl)phosphinic acid chloride), TBTU (O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), and the like. Examples of combinations of condensation agents and condensation additives include combinations of condensation agents such as DIC (diisopropylcarbodiimide), EDC·HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, WSC·HCl), and DCC (dicyclohexylcarbodiimide), and combinations of condensation additives such as HOAt (1-hydroxy-7-azabenzotriazole), HOCt (1-hydroxy-1H-1,2,3-triazole-5-carboxylic acid ethyl ester), HOBt (1-hydroxybenzotriazole), HOOBt (3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine), and DMAP (N,N-dimethyl-4-aminopyridine).

[0057] The reaction temperature for the condensation reaction in this step is not particularly limited as long as the reaction proceeds, but is preferably −10° C. to 60° C., and more preferably 5° C. to 35° C. The reaction time varies depending on the types of oligonucleotides, antioxidants, and solvents used, reaction temperature, etc., but is, for example, 5 minutes to 24 hours.

[0058] This step can be carried out, for example, by adding nucleic acid B and an antioxidant to a solution in which nucleic acid A has been dissolved, and then adding an activator, but this is one embodiment and the present invention is not limited to this embodiment.

[0059] In the present invention, in order to suppress the production of by-products during step (2), a step of quenching the activated nucleic acid B by the phosphoramidite method or the like can be further included prior to step (2).

[0060] As the quenching agent, those known in the art can be used as the quenching agent in this step. The quenching agent may be used alone or in combination of two or more. Examples of the quenching agent include alcohols (e.g., optionally halogenated monohydric alcohols such as methanol, 2-propanol, t-butanol, 2,2,2-trifluoroethanol (TFE), tetrahydrofurfuryl alcohol, furfuryl alcohol, 2,3-O-isopropylidene-D-ribofuranose, 3'-O-triisopropylsilyl-thymidine, etc., optionally halogenated polyhydric alcohols such as ethylene glycol and diethylene glycol, etc.), phenols (e.g., 4-nitrophenol, pentafluorophenol, etc.), and amines (e.g., morpholine, etc.).

[0061] The quenching agent is preferably at least one selected from alcohols and amines, more preferably at least one selected from methanol, 2-propanol, t-butanol, 2,2,2-trifluoroethanol, tetrahydrofurfuryl alcohol, and morpholine, and among these, 2,2,2-trifluoroethanol is preferred.

[0062] The amount of the quenching agent used in this step is preferably 1 to 20 mol, more preferably 1 to 15 mol, and even more preferably 1 to 10 mol, relative to 1 mol of the nucleic acid B used in step (1).

[0063] The temperature of the reaction solution after addition of the quenching agent is not particularly limited as long as it can quench nucleic acid B, but is preferably 5°C to 40°C, more preferably 15°C to 30°C.

[0064] Step (2) Sulfurization reaction using a sulfurizing agent

[0065] The sulfurizing agent used in this step is not particularly limited as long as it has the ability to convert a phosphite bond or a phosphorous diester bond to a phosphorothioate bond. Examples of the sulfurizing agent include 5-[(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazole-3-thione (DDTT), 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent), 3H-1,2-benzodithiol-3-one, phenylacetyl disulfide (PADS), tetrahydrofuran (THF), and the like. Preferred are tetraethylthiuram disulfide (TETD), dipentamethylene thiuram tetrasulfide, 5-phenyl-3H-1,2,4-dithiazol-3-one (POS), 3-amino-1,2,4-dithiazole-5-thione (ADTT, xanthan hydride), sulfur, cyanoethyl methylthiosulfonate, N-[(2-cyanoethyl)thio]succinimide, N-[(2-cyanoethyl)thio]morpholine-3,5-dione, and N-[(2-cyanoethyl)thio]phthalimide.

[0066] From the viewpoint of allowing a good reaction to proceed, 5-[(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazole-3-thione (DDTT), 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent), 3H-1,2-benzodithiol-3-one, phenylacetyl disulfide (PADS), 5-phenyl-3H-1,2,4-dithiazole-3-one (POS), cyanoethyl methylthiosulfonate, and N-[(2-cyanoethyl)thio]phthalimide are more preferred, and 5-[(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazole-3-thione is more preferred. Azole-3-thione (DDTT), 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent), 5-phenyl-3H-1,2,4-dithiazol-3-one (POS), cyanoethyl methylthiosulfonate, and N-[(2-cyanoethyl)thio]phthalimide are more preferred, and 5-[(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazol-3-one (DDTT), 5-phenyl-3H-1,2,4-dithiazol-3-one (POS), cyanoethyl methylthiosulfonate, and N-[(2-cyanoethyl)thio]phthalimide are particularly preferred. Such sulfurizing agents can be used by diluting with an appropriate solvent to a concentration of 0.05 to 2M. Such dilution solvents are not particularly limited as long as they are inert to the reaction, and examples thereof include dichloromethane, acetonitrile, pyridine, and any mixtures thereof.

[0067] The amount of the sulfurizing agent used is, for example, 1 to 50 mol, and preferably 1 to 5 mol, relative to 1 mol of the nucleic acid A used in step (1).

[0068] The reaction temperature is not particularly limited as long as the reaction proceeds, but is preferably 0° C. to 60° C., and more preferably 10° C. to 40° C. The reaction time varies depending on the type of phosphite or phosphorous diester, the type of sulfurizing agent used, the reaction temperature, etc., but is, for example, 1 minute to 3 hours.

[0069] The sulfurization reaction with a sulfurizing agent in step (2) may be carried out each time the condensation reaction in the presence of an antioxidant in step (1) is carried out, or may be carried out collectively after the nucleoside, nucleotide, or oligonucleotide is elongated by several condensation reactions in the presence of an antioxidant in step (1).Those skilled in the art can carry out the present invention by appropriately selecting the method according to the reaction conditions, such as the method for forming the phosphite or phosphorous diester used.

[0070] The above step (1) and / or step (2) can be carried out regardless of the presence or absence of a protecting group for the hydroxyl group at the 5' position of nucleic acid B. For example, the separation step described below can be carried out after step (1), or step (1) can be followed by step (2) and then by the separation step described below. Thus, in the nucleic acid elongation step, when the hydroxyl group at the 5' position of nucleic acid B is protected, deprotection of the protecting group is an optional step and can be carried out according to an appropriate synthesis plan.

[0071] Steps (1) and (2) of the present invention have been described in detail above. By carrying out the present invention, the drawbacks of the conventional method can be improved, and the desired phosphorothioate site can be introduced more efficiently into the internucleic acid bond of the oligonucleotide. The present invention is an excellent method for producing an oligonucleotide having a phosphorothioate site.

[0072] In carrying out the present invention, the following steps which are carried out as necessary in the production process of oligonucleotides in the art can also be carried out. In addition, in each step, the product may be isolated before carrying out the next step, or each step may be appropriately carried out consecutively.

[0073] [Deprotection process] In this step, the protecting group of the 5'-position hydroxyl group of the oligonucleotide having a phosphorothioate site obtained in step (2) is removed by acid to obtain an oligonucleotide having a phosphorothioate site with an unprotected 5'-position hydroxyl group. This step can be performed by a method commonly used in the art (see, for example, International Publication No. WO 2012 / 157723, International Publication No. WO 2013 / 122236, and International Publication No. WO 2017 / 104836).

[0074] [Neutralization process] After the deprotection step, a base may be added to the reaction solution to neutralize the acid used. However, in the production method of the present invention, the acid used in the deprotection step can be removed from the reaction system by performing the solid-liquid separation or extraction and washing described below, as necessary. Therefore, the neutralization step is not essential. This step can be performed by a method commonly used in the art (see, for example, International Publication No. WO 2012 / 157723, International Publication No. WO 2013 / 122236, International Publication No. WO 2017 / 104836).

[0075] [Solid-liquid separation or extraction process] In this step, when any group of the oligonucleotide having a phosphorothioate site in which the 5'-position hydroxyl group obtained above is not removed under acidic conditions but is protected with a protecting group (pseudo solid-phase protecting group) that is removable under basic conditions, a polar solvent is added to the reaction solution containing the oligonucleotide, and the oligonucleotide is precipitated and purified (solid-liquid separation), or a polar solvent is added to the reaction solution, the solution is separated between the polar solvent and the nonpolar solvent, and the oligonucleotide is transferred to the nonpolar solvent layer and purified (extraction). This step can be performed, for example, by the methods described in detail in WO 2012 / 157723, WO 2013 / 122236, and WO 2017 / 104836.

[0076] When the oligonucleotide having a phosphorothioate site obtained in step (2) has a nucleic acid sequence of the final interest, it can be subsequently subjected to a "deprotection step" and an "isolation step". That is, after removing all the protecting groups of the oligonucleotide having a phosphorothioate site obtained in step (2), a step of isolating the oligonucleotide having an unprotected phosphorothioate site can be further included. This step can be carried out, for example, by the method described in detail in WO 2012 / 157723, WO 2013 / 122236, and WO 2017 / 104836.

[0077] The progress of the reaction in each step can be confirmed by the same method as that used in general liquid phase organic synthesis reactions, i.e., the reaction can be tracked by thin layer silica gel chromatography, high performance liquid chromatography, etc.

[0078] The steps (1) and (2) of the present invention have been described in detail above, and a method for efficiently introducing a phosphorothioate site into a target site during the process of nucleic acid elongation has been described. The present invention is characterized in that, when introducing a phosphorothioate site, a condensation reaction is carried out between appropriately protected nucleic acid A and nucleic acid B in the presence of a specific antioxidant, and as long as this feature can be utilized, the production method of the present invention can be widely applied to appropriately protected nucleosides, nucleotides, or oligonucleotides (which may have phosphorothioate sites) regardless of the structure of the nucleic acid. The oligonucleotides produced by the present invention include not only oligonucleotides in which all phosphorus atoms are phosphorothioate sites modified with sulfur (All PS bodies), but also oligonucleotides in which only a specific portion of phosphorus atoms are phosphorothioate sites modified with sulfur, and the remaining phosphorus atoms are phosphates that are not modified with sulfur (Mixed Backbone Oligonucleotides (generally sometimes referred to as "MBOs")).

[0079] The nucleic acid A and the nucleic acid B to which the production method of the present invention is applied will be described in detail below.

[0080] In one embodiment in which the manufacturing method of the present invention is applied, Nucleic acid A is a nucleoside, nucleotide, or oligonucleotide in which the 5'-position hydroxyl group is not protected and other groups may be protected with a protecting group used in nucleic acid synthesis or may be bound to a solid phase support; Nucleic acid B is a nucleoside, nucleotide or oligonucleotide in which the 3'-position hydroxyl group or 3'-position amino group has been modified by a method for forming a phosphite or a phosphorous diester (e.g., a method selected from the phosphoramidite method, the H-phosphonate method, the dihalophosphine method, and the oxazaphospholidine method) and other groups may be protected with protecting groups used in nucleic acid synthesis.

[0081] In a more preferred embodiment herein, nucleic acid B is a nucleoside, nucleotide, or oligonucleotide in which the 3'-position hydroxyl group has been modified by a method for forming a phosphite or phosphite diester, and other groups may be protected with protecting groups used in nucleic acid synthesis.

[0082] As used herein, the term "nucleoside," which is a structural unit of an oligonucleotide, refers to a compound in which a nucleic acid base is bound to the 1-position of a sugar (e.g., 2-deoxyribose, ribose, 2-deoxyribose or ribose in which the 2- and 4-carbon atoms are bound by a divalent organic group, etc.) via N-glycosidation. In this specification, "sugar" also includes amino sugars in which the hydroxyl group is replaced with an amino group, ribose in which the 2-hydroxyl group is replaced with a halogen atom, and ribose having an intramolecular cross-linking structure (LNA; BNA).

[0083] Examples of 2-deoxyribose or ribose in which the 2- and 4-carbon atoms are linked via a divalent organic group include the following compounds. In the following formula, R represents a hydrogen atom, a substituted or unsubstituted hydrocarbon group, a substituted or unsubstituted hydroxyl group, or a substituted or unsubstituted amino group, and R' represents a hydrogen atom or a hydroxyl group.

[0084] [ka]

[0085] Examples of amino sugars include 2-deoxyribose in which the hydroxyl group at position 3 is replaced with an amino group, ribose in which the hydroxyl group at position 3 is replaced with an amino group, and ribose in which the hydroxyl group at position 3 is replaced with an amino group and the hydroxyl group at position 2 is replaced with a halogen (in the formula below, Xs represents a halogen atom).

[0086] [ka]

[0087] As used herein, the term "phosphate group" includes not only -OP(O)(OH)2 but also groups in which the oxygen atom is replaced by a sulfur atom or NH (e.g., -OP(S)(OH)2, -NH-P(O)(OH)2, -NH-P(S)(OH)2). The term "phosphate group" also includes groups in which the hydroxyl group (-OH) in a phosphate group is replaced by -ORp (wherein Rp represents an organic group such as a protecting group for a phosphate group) (e.g., a protected phosphate group).

[0088] In the present specification, the term "nucleotide" refers to a compound in which a phosphate group is bound to a nucleoside. Examples of nucleotides in which the 3'- or 5'-hydroxyl group is replaced with a phosphate group include compounds represented by the following formula (where R m1 and R m2 each independently represents a hydrogen atom or an organic group (excluding a nucleoside residue); X mrepresents a hydrogen atom, a hydroxyl group or a halogen atom, R1 and R2 represent a hydrogen atom or an alkyl group, R1 and R2 may be bonded to each other to form a 5- or 6-membered ring together with other atoms adjacent to them, and R3 and R4 represent a hydrogen atom or a phenyl group).

[0089] [ka]

[0090] [ka]

[0091] Other preferred examples of nucleotides in which the 3'- or 5'-hydroxyl group is replaced with a phosphate group include those represented by the following formulae (I'), (II') and (III').

[0092] [ka]

[0093] [In the above formulas (I'), (II') and (III'), * indicates the bond position with the 3'-hydroxyl group or 3'-amino group; L 7 indicates -O- or -S-; L 8 -LO-, -LS-, -LC(R 1 )(R 2 )-O-, -LC(R 1 )(R 2 )-S-, -L s -O- or -L s -S-indicated; Ring A is an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; Ring A' is an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and is represented by -N(R 6 )-moiety; L is a covalent bond or an optionally substituted C 1-6 represents alkylene, where one or more methylene units may be independently replaced by -L'-; Each L' is independently a covalent bond, an optionally substituted divalent C 1-3 Alkylene, -C(R 3 )(R 4 )-, -C(R 3 )(R 4 )-C(R 3 )(R 4 )-, -Cy- or -C(R 3 )[C(R 4 )3]-indicated; R 1 , R 2 , R 3 , R 4 and R 5 are independently hydrogen, -L s -R, halogen, -CN, -NO2, -L s -Si(R)3, -OR, -SR or -N(R)2; Each L s are independently a covalent bond or C 1-30 C having an aliphatic group and 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. 1-30 represents an optionally substituted divalent linear or branched radical selected from heteroaliphatic radicals, wherein one or more methylene units are independently selected from C 1-6 Alkylene, C 1-6alkenylene, -C≡C-, a divalent C1-C6 heteroaliphatic group having 1 to 5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S-, -C(O)O-, -P(O)(OR')-, -P(O)(SR')-, -P(O)(R')-, -P(O)(NR')-, -P( and one or more carbon atoms are optionally replaced by an optionally substituted group selected from Cy(OR')-, -P(S)(SR')-, -P(S)(R')-, -P(S)(NR')-, -P(R')-, -P(OR')-, -P(SR')-, -P(NR')-, -P(OR')[B(R')3]-, -OP(O)(OR')O-, -OP(O)(SR')O-, -OP(O)(R')O-, -OP(O)(NR')O-, -OP(OR')O-, -OP(SR')O-, -OP(NR')O-, -OP(R')O- and -OP(OR')[B(R')3]O-, and one or more carbon atoms are independently selected from Cy(OR')-, -P(S)(SR')-, -P(S)(R')-, -P(S)(NR')-, -P(R')-, -P(OR')-, -P(SR')-, -P(NR')O-, -OP(R')O- and -OP(OR')[B(R')3]O-. L may be replaced by; Each -Cy- is independently C 3-20 Alicyclic ring, C 6-20 represents an optionally substituted divalent group selected from an aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; Each Cy L is independently 3-20 Alicyclic ring, C 6-20 represents an optionally substituted tetravalent group selected from an aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; each R' independently represents -R, -C(O)R, -CO2R, or -SO2R; t indicates 0 to 20; R 6 denotes R'; R 7 indicates -OH or -SH; R 8 -LR 7 , -LC(R 1 )(R 2 )-R 7 or -L s -R 7 indicates; R 1 , R 2 , R 3 and R 4 At least one of is not hydrogen; Each R is independently hydrogen or C 1-30 C having 1 to 10 heteroatoms independently selected from aliphatic groups, oxygen, nitrogen, sulfur, phosphorus, and silicon 1-30 Heteroaliphatic groups, C 6-30 Aryl group, C 6-30 C having 1 to 10 heteroatoms independently selected from arylaliphatic groups, oxygen, nitrogen, sulfur, phosphorus, and silicon 6-30 an optionally substituted group selected from an arylheteroaliphatic group, a 5-30 membered heteroaryl group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-30 membered heterocyclyl group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups may independently join together to form a covalent bond; or two or more R on the same atom may independently, together with the atom, form an optionally substituted 3-30 membered monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or Two or more R groups on two or more atoms may independently, together with the intervening atoms, form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. However, R 1 and R 2 are not the same, R 3 and R 4 are not the same.]

[0094] In the present specification, the term "oligonucleotide" refers to a compound in which one or more nucleotides are linked to a nucleoside. Unless otherwise specified, the term "oligonucleotide" also includes a phosphorothioated oligonucleotide in which the oxygen atom of the phosphate group is replaced with a sulfur atom, an oligonucleotide in which the -O- of the phosphate group is replaced with -NH-, and an oligonucleotide in which the hydroxyl group (-OH) in the phosphate group is replaced with -ORp (wherein Rp represents an organic group). The number of nucleosides in the oligonucleotide of the present invention is not particularly limited, but is preferably 3 to 50, more preferably 5 to 30.

[0095] As used herein, the term "3'-position amino group" refers to an amino group bound to the 3'-position carbon atom of a nucleoside, nucleotide or oligonucleotide. As used herein, the term "5' amino group" refers to an amino group bound to the 5' carbon atom of a nucleoside, nucleotide or oligonucleotide.

[0096] As used herein, the term "3' phosphate group" refers to a phosphate group bound to the 3' carbon atom of a nucleotide or oligonucleotide. As used herein, the term "5' phosphate group" refers to a phosphate group bound to the 5' carbon atom of a nucleotide or oligonucleotide.

[0097] In the present specification, the term "nucleic acid base" is not particularly limited as long as it is used in the synthesis of nucleic acid, and examples thereof include pyrimidine bases such as cytosyl, uracil, and thyminyl groups, and purine bases such as adenyl and guanyl groups. In addition to the above-mentioned groups, the nucleic acid base also includes modified nucleic acid bases (e.g., 8-bromoadenylation group, 8-bromoguanyl group, 5-bromocytosyl group, 5-iodocytosyl group, 5-bromouracil group, 5-iodouracil group, 5-fluorouracil group, 5-methylcytosyl group, 8-oxoguanyl group, hypoxanthinyl group, etc.) in which the nucleic acid base is substituted with 1 to 3 optional substituents (e.g., halogen atom, alkyl group, aralkyl group, alkoxy group, acyl group, alkoxyalkyl group, hydroxyl group, amino group, monoalkylamino, dialkylamino, carboxy, cyano, nitro, etc.) at any position. Furthermore, the term "optionally protected nucleobase" means, for example, that in an adenyl group, a guanyl group, or a cytosyl group, which is a nucleobase having an amino group, the amino group may be protected by an "amino group-protecting group" described below, and a nucleobase in which the amino group is protected by a protecting group that can withstand the deprotection conditions for the 5'-position of a nucleotide is preferred.

[0098] As used herein, the "protecting group used in nucleic acid synthesis" is not particularly limited as long as it is one that is commonly used in the field of nucleic acid synthesis, and examples thereof include the following:

[0099] 1) Hydroxyl-protecting group (the hydroxyl-protecting group of the nucleic acid base portion is described below in the section on protecting groups of the nucleic acid base portion) Unless otherwise specified, the "hydroxyl-protecting group" is not particularly limited, and may be, for example, any protecting group described in PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd Edition, John Wiley & Sons (1999) or the like. Specifically, examples of the protecting group include a methyl group, a benzyl group, a p-methoxybenzyl group, a tert-butyl group, a methoxymethyl group, a methoxyethyl group, a 2-tetrahydropyranyl group, an ethoxyethyl group, a cyanoethyl group, a cyanoethoxymethyl group, a phenylcarbamoyl group, a 1,1-dioxothiomorpholine-4-thiocarbamoyl group, an acetyl group, a pivaloyl group, a benzoyl group, a trimethylsilyl group, a triethylsilyl group, a triisopropylsilyl group, a tert-butyldimethylsilyl group, a [(triisopropylsilyl)oxy]methyl (Tom) group, and a 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) group. The protecting group for the hydroxyl group, which may be protected, is preferably a triethylsilyl group, a triisopropylsilyl group, or a tert-butyldimethylsilyl group, and is particularly preferably a tert-butyldimethylsilyl group from the viewpoints of economy and availability.

[0100] 2) Amino protecting group Unless otherwise specified, the "protective group for amino group" is not particularly limited, and examples thereof include the protective groups described in Greene's PROTECTIVE GROUPS in ORGANIC SYNTHESIS, 4th Edition, published by Wiley-Interscience (2006), etc. Specific examples of the protective group include a pivaloyl group, a pivaloyloxymethyl group, an acetyl group, a trifluoroacetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a benzoyl group, an isobutyryl group, a (2-hexyl)decanoyl group, a dimethylformamidinyl group, a 1-(dimethylamino)ethylidene group, and a 9-fluorenylmethyloxycarbonyl group. Of these, acetyl group, phenoxyacetyl group, 4-isopropylphenoxyacetyl group, benzoyl group, isobutyryl group, (2-hexyl)decanoyl group, dimethylformamidinyl group, and 1-(dimethylamino)ethylidene group are preferred.

[0101] 3) Protecting groups for nucleic acid bases In the adenyl group, guanyl group, or cytosyl group, which is a nucleic acid base having an amino group, the amino group may be protected by the above-mentioned "amino group protecting group". A nucleic acid base in which the amino group of the nucleic acid base is protected by a protecting group that can withstand the deprotection conditions of the 5' position of a nucleotide is preferred. The carbonyl group of the nucleic acid base may also be protected. For example, phenol, 2,5-dichlorophenol, 3-chlorophenol, 3,5-dichlorophenol, 2-formylphenol, 2-naphthol, 4-methoxyphenol, 4-chlorophenol, 2-nitrophenol, 4-nitrophenol, 4-acetylaminophenol, pentafluorophenol, 4-pivaloyloxybenzyl alcohol, 4-nitrophenethyl alcohol, 2-(methylsulfonyl)ethanol, 2-(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol, dimethylcarbamic acid chloride, diethylcarbamic acid chloride, ethylphenylcarbamic acid chloride, 1-pyrrolidine carboxylic acid chloride, 4-morpholine carboxylic acid chloride, diphenylcarbamic acid chloride, etc. may be reacted to protect the carbonyl group of the nucleic acid base. Here, there are cases where it is not necessary to introduce a protective group for the carbonyl group.

[0102] A more preferred embodiment in which the production method of the present invention is applied is as follows: Nucleic acid A is a nucleoside, nucleotide or oligonucleotide in which the 5'-position hydroxyl group is not protected, and at least one group selected from the amino group and imino group of the nucleic acid base, the 2'-position hydroxyl group, the 3'-position hydroxyl group and the 3'-position amino group of the ribose residue, and the 3'-position hydroxyl group and the 3'-position amino group of the deoxyribose residue is protected with a protecting group (pseudo solid-phase protecting group) that is not removable under acidic conditions and is removable under basic conditions, and other groups may be further protected with a protecting group used in nucleic acid synthesis (hereinafter sometimes referred to as "nucleic acid a"); a nucleoside, nucleotide or oligonucleotide (hereinafter sometimes referred to as "nucleic acid α") in which the 5'-position hydroxyl group is not protected, one OH of the phosphate group at the 3'-position terminal is replaced with -OLn1-OH (wherein Ln1 represents an organic group), the hydroxyl group of -OLn1-OH is protected with a protecting group (pseudo solid-phase protecting group) that is not removable under acidic conditions but is removable under basic conditions, and other groups may be further protected with protecting groups used in nucleic acid synthesis; Nucleic acid B is a nucleoside, nucleotide or oligonucleotide in which the 3'-position hydroxyl group or the 3'-position amino group has been modified by a method selected from the phosphoramidite method, the H-phosphonate method, the dihalophosphine method and the oxazaphospholidine method, the 5'-position hydroxyl group is protected with a temporary protecting group removable under acidic conditions, and other groups may be further protected with protecting groups selected from protecting groups that are not removable under acidic conditions but are removable under basic conditions (pseudo solid-phase protecting groups) and protecting groups used in nucleic acid synthesis (hereinafter, sometimes referred to as "nucleic acid b").

[0103] Examples of the "protecting group used in nucleic acid synthesis" in the nucleic acid a, the nucleic acid α, and the nucleic acid b include the same protecting groups as those explained above for the nucleic acid A and the nucleic acid B.

[0104] The "protective group (pseudo solid-phase protecting group) that is not removed under acidic conditions and is removable under basic conditions" in nucleic acid a, nucleic acid α, and nucleic acid b can each independently be a protecting group having an organic group with a linear aliphatic hydrocarbon group having 10 or more carbon atoms or at least one aliphatic hydrocarbon group having one or more branched chains, and a total carbon number of 14 to 300. The pseudo solid-phase protecting group imparts hydrophobicity to nucleic acid a, nucleic acid α, nucleic acid b, and the oligonucleotide having a phosphorothioate site obtained by steps (1) and (2), thereby improving the solubility in a nonpolar solvent. It can also reduce the solubility in a polar solvent. The nucleoside, nucleotide, or oligonucleotide protected by such a pseudo solid-phase protecting group can undergo a condensation reaction even in the liquid phase of a nonpolar solvent (step (1)), and by adding a polar solvent to the reaction solution obtained in step (2), the oligonucleotide having a phosphorothioate site protected by the pseudo solid-phase protecting group precipitates, and solid-liquid separation can be performed. Alternatively, a polar solvent is added to the reaction solution obtained in step (2), and the reaction solution is partitioned between the polar solvent and the non-polar solvent, and the oligonucleotide having the phosphorothioate moiety is transferred to the non-polar solvent, whereby the oligonucleotide can be extracted. As such a pseudo-solid-phase protecting group, for example, those described in International Publication No. 2012 / 157723, International Publication No. 2013 / 122236, International Publication No. 2017 / 104836, International Publication No. 2013 / 179412, International Publication No. 2014 / 077292, and International Publication No. 2017 / 086397 can be used.

[0105] The "protecting group used in nucleic acid synthesis" is not particularly limited as long as it is one that is commonly used in the field of nucleic acid synthesis, and includes the above-mentioned low molecular weight protecting groups. However, when purification by solid-liquid separation is performed after step (2), the pseudo-solid-phase protecting group is preferably a protecting group having a linear aliphatic hydrocarbon group having 10 or more carbon atoms, and when purification by extraction is performed after step (2), the pseudo-solid-phase protecting group is preferably a protecting group having at least one aliphatic hydrocarbon group having one or more branches and having an organic group with a total carbon number of 14 to 300.

[0106] First, a pseudo-solid-phase protecting group suitable for solid-liquid separation will be described. Examples of pseudo-solid-phase protecting groups suitable for solid-liquid separation include C 10 or more linear aliphatic hydrocarbon groups bonded via a single bond or a linker, and C 10 or more linear aliphatic hydrocarbon groups bonded via a linker. 6-14 Examples of the protecting group include a protecting group having a hydrocarbon ring.

[0107] The linear aliphatic hydrocarbon group having 10 or more carbon atoms is preferably a linear C 10-40 Alkyl groups and linear C 10-40 alkenyl groups, more preferably linear C 10-40 An alkyl group, more preferably a linear C 10-30 The alkyl group is preferably a straight-chain C 12-28 is an alkyl group, most preferably a linear C 14-26 It is an alkyl group.

[0108] The linker is preferably -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH-, -NHC(=O)-, -S-, -SO-, -SO2-, and -Si(R')(R")O-, -Si(R')(R")- (R' and R" are each independently a hydrogen atom or C 1-22 represents a hydrocarbon group.) and more preferably selected from -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- and -NHC(=O)-, and more preferably -O-.

[0109] Said C 6-14 The hydrocarbon ring is preferably selected from a benzene ring, a naphthalene ring, and a cyclohexane ring, more preferably selected from a benzene ring and a cyclohexane ring, and further preferably a benzene ring.

[0110] The pseudo-solid-phase protecting groups suitable for solid-liquid separation are preferably linear C 10-40 It is a protecting group having a benzene ring to which an alkyl group is bonded via a single bond or -O- and a hydrocarbon group is bonded via -O-.

[0111] Next, a pseudo-solid-phase protecting group suitable for extraction will be described. The "branched chain" in the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains and having a total carbon number of 14 to 300" is a linear or branched saturated aliphatic hydrocarbon group, and 1-6 Alkyl groups are preferred, C 1-4 An alkyl group is more preferred, and a methyl group or an ethyl group is even more preferred. The "branched chain" may also be substituted with one or more halogen atoms.

[0112] The "aliphatic hydrocarbon group" in the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains and a total carbon number of 14 to 300" is a linear, saturated or unsaturated aliphatic hydrocarbon group, 2-300 Alkyl groups (preferably C 3-100 Alkyl groups, more preferably C 3-60 Alkyl group), C 2-300 Alkenyl groups (preferably C 3-100 Alkenyl groups, more preferably C 3-60 alkenyl group) or C 2-300 Alkynyl groups (preferably C 3-100 Alkynyl groups, more preferably C 3-60 alkynyl group).

[0113] The position of the "aliphatic hydrocarbon group having one or more branched chains" in the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains and having a total carbon number of 14 to 300" is not particularly limited, and may be present at a terminal (monovalent group) or at another position (for example, a divalent group).

[0114] Examples of the "aliphatic hydrocarbon group having one or more branched chains" include branched isomers such as propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group (lauryl group), tridecyl group, myristyl group, cetyl group, stearyl group, arachyl group, behenyl group, oleyl group, linolyl group, and lignoceryl group, and include monovalent groups having one or more branched chains and divalent groups derived therefrom. The "aliphatic hydrocarbon group having one or more branched chains" is preferably 3,7,11-trimethyldodecyl group, 3,7,11,15-tetramethylhexadecyl group (hereinafter sometimes referred to as 2,3-dihydrophytyl group), 2,2,4,8,10,10-hexamethylundecane-5-yl group, etc.

[0115] When there are multiple "aliphatic hydrocarbon groups having one or more branched chains" in the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains and having a total carbon number of 14 to 300", each of the "aliphatic hydrocarbon groups having one or more branched chains" may be the same or different.

[0116] In the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains and a total carbon number of 14 to 300", the moiety other than the "aliphatic hydrocarbon group having one or more branched chains" can be set arbitrarily. For example, it may have a moiety such as -O-, -S-, -CO-, -NH-, -COO-, -OCONH-, -CONH-, -NHCO-, or a hydrocarbon group (monovalent or divalent group). Examples of the "hydrocarbon group" include an aliphatic hydrocarbon group, an aromatic aliphatic hydrocarbon group, a monocyclic saturated hydrocarbon group, and an aromatic hydrocarbon group. Specifically, for example, monovalent groups such as an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, and an aralkyl group, and divalent groups derived therefrom, are used. Examples of the "alkyl group" include C 1-6 Alkyl groups are preferred, and examples thereof include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like. 2-6Alkenyl groups are preferred, and examples thereof include vinyl, 1-propenyl, allyl, isopropenyl, butenyl, and isobutenyl. The "alkynyl group" is C 2-6 Alkynyl groups are preferred, such as ethynyl, propargyl, 1-propynyl, etc. "Cycloalkyl groups" include C 3-6 Cycloalkyl groups are preferred, such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. 6-14 Aryl groups are preferred, and examples thereof include phenyl, 1-naphthyl, 2-naphthyl, biphenylyl, and 2-anthryl. 6-10 An aryl group is more preferred, and a phenyl group is particularly preferred. 7-20 Aralkyl groups are preferred, and examples thereof include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-naphthylethyl, and 1-naphthylpropyl. Among these, C 7-16 Aralkyl group (C 6-10 Aryl-C 1-6 An alkyl group is more preferable, and benzyl is particularly preferable. The "hydrocarbon group" may be substituted with a substituent selected from a halogen atom (a chlorine atom, a bromine atom, a fluorine atom, an iodine atom), an oxo group, and the like.

[0117] The "total number of carbon atoms" in the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains and a total carbon number of 14 to 300" is 14 or more, preferably 16 or more, more preferably 18 or more, and 300 or less, preferably 200 or less, more preferably 160 or less. The number of branched chains in the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains and a total carbon number of 14 to 300" is not particularly limited, but is preferably 2 or more, more preferably 3 or more, more preferably 4 or more, more preferably 8 or more, and even more preferably 10 or more. The more the number of branched chains, the better the nucleoside or oligonucleotide protected by a pseudo-solid-phase protecting group having an organic group having at least one aliphatic hydrocarbon group having one or more branched chains and a total carbon number of 14 to 300 is dissolved in an organic solvent (particularly a non-polar solvent) even when the oligonucleotide chain becomes a long chain.

[0118] As the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains and having a total carbon number of 14 to 300", there may be mentioned the organic group represented by the formula (A):

[0119] [ka]

[0120] [In the formula, * indicates the bond position to the adjacent atom; R 14 and R 15 are each independently a hydrogen atom or C 1-4 represents an alkyl group; and X 1 is a single bond or C 1-4 Represents an alkylene group. However, R 14 and R 15 cannot both be hydrogen atoms. Preferred are groups having the same or different divalent groups represented by the following formula:

[0121] Examples of groups having a divalent group represented by formula (A) include groups represented by any of the following formulas (B) to (D). Note that the number of carbon atoms, the number of repeating units (m1, n0 to n2), etc. in the definitions of each symbol in formulas (B) to (D) are shown for convenience, and can be appropriately changed within the above definitions so that the total number of carbon atoms is 14 or more (preferably 16 or more, more preferably 18 or more) and 300 or less (preferably 200 or less, more preferably 160 or less). Formulas (B) to (D) will be explained in order below.

[0122] Formula (B) is as follows:

[0123] [ka]

[0124] [In the formula, * indicates the bond position to the adjacent atom; R 16 and R 17 represent a hydrogen atom or together represent =O; n0 represents an integer from 2 to 40; n0 R 18 and R 19 are each independently a hydrogen atom or C 1-4 represents an alkyl group; n0 X's 2 are each independently a single bond or C 1-4 Represents an alkylene group; R 20 is a hydrogen atom or C 1-4 represents an alkyl group; and R 21 is C 1-4 Indicates an alkyl group. However, R 18 and R 19 are not both hydrogen atoms and n0 is 2, R 20 is C 1-4 represents an alkyl group.

[0125] The group of formula (B) is R 16 and R 17 are both hydrogen atoms; n0 is an integer from 2 to 40; n0 R 18 and R 19 are each independently a hydrogen atom, a methyl group, or an ethyl group; n0 X's 2 are each independently a single bond, a methylene group, or an ethylene group; and R 20 is a hydrogen atom, a methyl group, or an ethyl group Group (wherein R 18 and R 19 are not both hydrogen atoms and n0 is 2, R 20 represents a methyl or ethyl group.) is preferred.

[0126] More preferred groups of formula (B) are branched isomers having 14 to 160 carbon atoms, such as myristyl, cetyl, stearyl, arachyl and behenyl groups, with 2,3-dihydrophytyl, 3,7,11-trimethyldodecyl and 2,2,4,8,10,10-hexamethyl-5-dodecanoyl being particularly preferred.

[0127] Formula (C) is as follows:

[0128] [ka]

[0129] [In the formula, * indicates the bond position to the adjacent atom; m1 OR 22 each independently represents a hydroxyl group substituted with a group represented by formula (B); and m1 represents an integer of 1 to 3. In addition, the "group represented by formula (B)" in formula (C) is as described above, except that the * indicates the bonding position to O (that is, the adjacent atom).

[0130] In the group of formula (C), R22 is more preferably a branched isomer having 14 to 30 carbon atoms, such as a myristyl group, a cetyl group, a stearyl group, an arachyl group, or a behenyl group, and among these, a 2,3-dihydrophytyl group or a 3,7,11-trimethyldodecyl group is particularly preferred.

[0131] Formula (D) is as follows:

[0132] [ka]

[0133] [In the formula, * indicates the bond position with Q; n1 represents an integer from 1 to 10; n2 represents an integer from 1 to 10; n1 R 26 and R 27 are each independently a hydrogen atom or C 1-4 represents an alkyl group; n1 X's 3 are each independently a single bond or C 1-4 Represents an alkylene group; n2 R 28 and R 29 are each independently a hydrogen atom or C 1-4 represents an alkyl group; n2 X's 5 are each independently a single bond or C 1-4 Represents an alkylene group; X 4 is a single bond or C 1-4 represents an alkylene group; and R 23 , R 24 , R 25 , R 30 , R 31 and R 32 are each independently a hydrogen atom or C 1-4 Indicates an alkyl group. However, R 26 and R 27 , and / or R 28 and R 29are not both hydrogen atoms and n1+n2 is 2, R 23 , R 24 and R 25 Two or more of the following are independently C 1-4 Indicates an alkyl group or R 30 , R 31 and R 32 Two or more of the following are independently C 1-4 represents an alkyl group.

[0134] The group of formula (D) is n1 is an integer from 1 to 5; n2 is an integer from 1 to 5; n1 R 26 and R 27 are each independently a hydrogen atom, a methyl group, or an ethyl group; n1 X's 3 are each independently a single bond, a methylene group, or an ethylene group; n2 R 28 and R 29 are each independently a hydrogen atom, a methyl group, or an ethyl group; n2 X's 5 are each independently a single bond, a methylene group, or an ethylene group; X 4 is a single bond, a methylene group or an ethylene group; and R 23 , R 24 , R 25 , R 30 , R 31 and R 32 are each independently a hydrogen atom or C 1-4 is an alkyl group Group (wherein R 26 and R 27 , and / or R 28 and R 29 are not both hydrogen atoms and n1+n2 is 2, R 23 , R 24 and R 25 Two or more of the following are independently C 1-4 Indicates an alkyl group or R30 , R 31 and R 32 Two or more of the following are independently C 1-4 It is more preferable that the alkyl group is an alkyl group.

[0135] Particularly preferred groups of formula (D) include n1 is an integer from 1 to 5; n2 is an integer from 1 to 5; n1 R 26 and R 27 are each independently a hydrogen atom or a methyl group; n1 X's 3 are each independently a single bond or a methylene group; n2 R 28 and R 29 are each independently a hydrogen atom or a methyl group; n2 X's 5 are each independently a single bond or a methylene group; X 4 is a single bond or a methylene group; and R 23 , R 24 , R 25 , R 31 , R 31 and R 32 is a methyl group Group (wherein R 26 and R 27 , and / or R 28 and R 29 However, neither of them are hydrogen atoms.

[0136] Specific examples of "an organic group having at least one aliphatic hydrocarbon group having one or more branched chains and a total carbon number of 14 to 300" include the following groups. In each group, * indicates a bonding position; n3 in the formula indicates an integer of 3 or more; and n4 can be appropriately set so that the total carbon number of the group is 14 to 300.

[0137] [ka]

[0138] Preferred specific examples of the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains and a total carbon number of 14 to 300" include the following groups: 3,7,11,15-Tetramethylhexadecyl group (also known as 2,3-dihydrophytyl group); 3,7,11-trimethyldodecyl group; 2,2,4,8,10,10-Hexamethyl-5-dodecanoyl group; 3,4,5-tri(3',7',11',15'-tetramethylhexadecyloxy)benzyl group; and 3,5-Di(3',7',11',15'-tetramethylhexadecyloxy)benzyl group.

[0139] The pseudo solid-phase protecting group is more preferably a group represented by the following formula (gI) (hereinafter sometimes abbreviated as "pseudo solid-phase protecting group (gI)"). **LYZ (gI) [In the formula, ** indicates the position of bond to the protecting group; L is a single bond or a group represented by formula (a1) or (a1′):

[0140] [ka]

[0141] (In the formula, * indicates the position of bond to Y; ** is as defined above; R 1 and R 2 are each independently 1-22 represents a hydrocarbon group; L 1 is an optionally substituted divalent C 1-22 represents a hydrocarbon group; L 2 indicates a single bond or ***C(=O)OR 4 -N(R 5)**** or ***C(=O)N(R 3 )-R 4 -N(R 5 )**** (wherein *** is L 1 indicates the bond position with C=O, and R 4 is C 1-22 R represents an alkylene group. 3 and R 5 are each independently a hydrogen atom or C 1-22 Indicates an alkyl group or R 3 and R 5 may be taken together to form a ring. Y represents a single bond, an oxygen atom, or NR (R represents a hydrogen atom, an alkyl group, or an aralkyl group); and Z is a formula (a2), (a2') or (a2"):

[0142] [ka]

[0143] [In the formula, * indicates bond position; R 6 is a hydrogen atom or R b is a group represented by the following formula (a3), R 6 is R 8 may be taken together with a single bond or -O- to form a condensed ring with ring A or ring B and ring C; k represents an integer of 1 to 4; k Q's each independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, or -NR 7 -, -C(=O)NH- or -NHC(=O)-; k R 7each independently represents a hydrocarbon group in which a linear aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linker, or represents an organic group having at least one aliphatic hydrocarbon group having one or more branched chains and having a total carbon number of 14 to 300; Ring A and ring B each independently have k QR 7 In addition to the above, halogen atoms and C optionally substituted with halogen atoms 1-6 C optionally substituted with alkyl and halogen atoms 1-6 may have a substituent selected from the group consisting of alkoxy groups; R a indicates a hydrogen atom; and R b is a hydrogen atom, or a group represented by the formula (a3):

[0144] [ka]

[0145] (In the formula, * indicates bond position; j represents an integer of 0 to 4; j Q's each independently have the same meaning as defined above; j R 9 each independently represents a hydrocarbon group in which a linear aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linker, or represents an organic group having at least one aliphatic hydrocarbon group having one or more branched chains and having a total carbon number of 14 to 300; R 8 represents a hydrogen atom or R 6 may be taken together with a single bond or -O- to form a condensed ring with ring A or ring B and ring C; and A ring C has j QR 9 In addition to the above, halogen atoms and C optionally substituted with halogen atoms 1-6 C optionally substituted with alkyl and halogen atoms 1-6) which may have a substituent selected from the group consisting of alkoxy groups; R a and R b are taken together to form an oxo group.

[0146] R in formula (a2), formula (a2') and formula (a2") 7 , and R in formula (a3) 9 The linear aliphatic hydrocarbon groups having 10 or more carbon atoms are preferably linear C 10-40 Alkyl groups and linear C 10-40 alkenyl groups, more preferably linear C 10-40 An alkyl group, more preferably a linear C 10-30 The alkyl group is preferably a straight-chain C 12-28 is an alkyl group, most preferably a linear C 14-26 It is an alkyl group. R in formula (a2), formula (a2') and formula (a2") 7 , and R in formula (a3) 9 Each linker contained in each of the formulas is preferably -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-, and more preferably -O-.

[0147] R in formula (a2), formula (a2') and formula (a2") 7 , and R in formula (a3) 9 The "hydrocarbon group having 10 or more carbon atoms and a straight-chain aliphatic hydrocarbon group bonded via a single bond or a linker" is preferably a straight-chain C 10-40 Alkyl group, 1-3 straight chain C 10-40 A benzyl group with an alkyl group bonded via -O-, or 1 to 3 straight-chain C 10-40 It is a cyclohexylmethyl group in which the alkyl group is bonded via -O-.

[0148] R in formula (a2), formula (a2') and formula (a2") 7 , and R in formula (a3)9 The "organic group having at least one aliphatic hydrocarbon group having one or more branched chains and having a total carbon number of 14 to 300" which is one embodiment of the above is preferably a group having a divalent group represented by the above formula (A), more preferably a group represented by any of the above formulas (B) to (D), even more preferably a group represented by the above formula (B), and particularly preferably a 2,3-dihydrophytyl group, a 3,7,11-trimethyldodecyl group, or a 2,2,4,8,10,10-hexamethyl-5-dodecanoyl group.

[0149] Q in formula (a2), formula (a2'), formula (a2") and formula (a3) ​​is preferably -O-, -C(=O)NH- or -NHC(=O)-, and more preferably -O-.

[0150] In formula (gI), a preferred embodiment of L represented by formula (a1) is L 1 But divalent C 1-22 a hydrocarbon group (wherein one or more -CH2- constituting the hydrocarbon group may be substituted with -O-, -C(=O)-, etc.), or CH2-O-1,4-phenylene-O-CH2; and L 2 is a single bond or ***C(=O)N(R 3 )-R 4 -N(R 5 )**** (wherein *** is L 1 indicates the bond position with C=O, and R 4 is C 1-6 R represents an alkylene group. 3 and R 5 each independently represents a hydrogen atom or an optionally substituted C 1-6 Indicates an alkyl group or R 3 and R 5 together with optionally substituted C 1-6 An alkylene group may be formed. It is based on

[0151] Another preferred embodiment of L represented by formula (a1) is L 1 But divalent C 1-22 is a hydrocarbon group; and L 2 is a single bond, It is based on

[0152] Another preferred embodiment of L represented by formula (a1) is L 1 is an ethylene group; and L 2 However, ***C(=O)N(R 3 )-R 4 -N(R 5 )**** (wherein *** is L 1 indicates the bond position with C=O, and R 4 is C 1-22 R represents an alkylene group. 3 and R 5 are each independently a hydrogen atom or C 1-22 Indicates an alkyl group or R 3 and R 5 may be taken together to form a ring. It is based on

[0153] Another preferred embodiment of L represented by formula (a1) is L 1 is an ethylene group; and L 2 However, ***C(=O)N(R 3 )-R 4 -N(R 5 )**** (wherein *** is L 1 indicates the bond position with C=O, and N(R 3 )-R 4 -N(R 5 ) portion forms a 1,4-piperazinediyl group. It is based on

[0154] Another preferred embodiment of L represented by formula (a1) is L 1 is an ethylene group; and L 2 However, ***C(=O)N(R 3 )-R 4 -N(R 5 )**** (wherein *** is L 1 indicates the bond position with C=O, and R 4 represents a pentylene group or a hexylene group, and R 3 and R 5 each independently represents a hydrogen atom or a methyl group; It is based on

[0155] A particularly preferred embodiment of L in formula (a1) is a succinyl group, which is easily available and inexpensive.

[0156] Next, L represented by formula (a1') in formula (gI) will be described. L in formula (a1') 1 is preferably a divalent C 6-10 It is an aromatic hydrocarbon group, and more preferably a phenylene group. L in formula (a1') 2 is preferably a single bond. L in formula (a1') 1 and L 2 A preferred combination of L 1 is divalent C 6-10 An aromatic hydrocarbon group, L 2 is a single bond. 1 and L 2 A more preferred combination of L 1 is a phenylene group, and L 2 is a single bond. R in formula (a1') 1 and R 2 are each independently preferably C 1-22 is an alkyl group, more preferably C 1-10 It is an alkyl group.

[0157] A preferred embodiment of L represented by formula (a1') is R 1 and R 2 However, each independently, C 1-22 is an alkyl group; L 1 But divalent C 6-10 is an aromatic hydrocarbon group; and L 2 is a single bond, It is based on

[0158] Another preferred embodiment of L represented by formula (a1') is R 1 and R 2 However, each independently, C 1-10 is an alkyl group; L 1 is a phenylene group; and L 2 is a single bond, It is based on

[0159] When Y in formula (gI) is NR, the R is preferably a hydrogen atom, C 1-6 Alkyl group or C 7-16 Y is an aralkyl group, more preferably a hydrogen atom, methyl, ethyl or benzyl, and further preferably a hydrogen atom. Y is preferably a single bond, an oxygen atom or NR, and more preferably a single bond or an oxygen atom.

[0160] Z in formula (gI) is preferably a group represented by formula (a2) or formula (a2"), more preferably a group represented by formula (a2"). Compared with a pseudo solid-phase protecting group having Z represented by formula (a2) (i.e., a benzyl group structure), the use of a pseudo solid-phase protecting group having Z represented by formula (a2") (i.e., a cyclohexylmethyl group structure) can dramatically improve the solubility of nucleosides, nucleotides, oligonucleotides (a), etc. in non-polar solvents. This allows the production method of the present invention to be carried out at a higher concentration, greatly improving productivity.

[0161] In formula (a2), R 6 is preferably a hydrogen atom. a and R b are preferably a hydrogen atom or together form an oxo group.

[0162] A preferred embodiment of Z represented by formula (a2) for solid-liquid separation is R a and R b is a hydrogen atom; R 6 is a hydrogen atom; k is an integer from 1 to 3; k Q's are -O-; and k R 7 However, each independently has a linear C 10-40 is an alkyl group, It is based on

[0163] Another preferred embodiment of Z represented by formula (a2) for solid-liquid separation is R a and R b is a hydrogen atom; R 6 is a hydrogen atom; k is an integer from 1 to 3; k Q's are -O-; k R 7 Each independently has 1 to 3 linear C 10-40 A benzyl group with an alkyl group bonded via -O-, or 1 to 3 straight-chain C 10-40 is a cyclohexylmethyl group in which the alkyl group is bonded through -O-; and A ring A has k QR 7 In addition to the above, halogen atoms and C optionally substituted with halogen atoms 1-6 C optionally substituted with alkyl and halogen atoms 1-6 may have a substituent selected from the group consisting of alkoxy groups; It is based on

[0164] Another preferred embodiment of Z represented by formula (a2) for solid-liquid separation is R a is a hydrogen atom; and R b Formula (a3) ​​(wherein * is a bonding position, j is an integer of 0 to 3, j Q's are -O-, and j R 9 However, each independently has a linear C 10-40 is an alkyl group, R 6 and R 8 are both hydrogen atoms. It is based on

[0165] Another preferred embodiment of Z represented by formula (a2) for solid-liquid separation is R a is a hydrogen atom; and R b Formula (a3) ​​(wherein * is a bonding position, j is an integer of 0 to 3, j Q's are -O-, and j R 9 However, each independently has a linear C 10-40 is an alkyl group, R 8 is R 6 and together with the ring A and the ring C, form a condensed ring. It is based on

[0166] Another preferred embodiment of Z represented by formula (a2) for solid-liquid separation is R a and R b together form an oxo group; R 6 is a hydrogen atom; k is an integer from 1 to 3; k Q's are -O-; and k R 7 However, each independently has a linear C 10-40 is an alkyl group, It is based on

[0167] Another preferred embodiment of Z represented by formula (a2) for solid-liquid separation is R a and R b together form an oxo group; R 6 is a hydrogen atom; k is an integer from 1 to 3; k Q's are -O-; k R 7 Each independently has 1 to 3 linear C 10-40 A benzyl group with an alkyl group bonded via -O-, or 1 to 3 straight-chain C 10-40 is a cyclohexylmethyl group in which the alkyl group is bonded through -O-; and A ring A has k QR 7 In addition to the above, halogen atoms and C optionally substituted with halogen atoms 1-6 C optionally substituted with alkyl and halogen atoms 1-6 may have a substituent selected from the group consisting of alkoxy groups; It is based on

[0168] In formula (a2"), R 6 is preferably a hydrogen atom. In addition, in formula (a2"), R a and R b are preferably a hydrogen atom or together form an oxo group.

[0169] A preferred embodiment of Z represented by the formula (a2") for solid-liquid separation is R a and R b is a hydrogen atom; R 6 is a hydrogen atom; k is an integer from 1 to 3; k Q's are -O-; and k R 7 However, each independently has a linear C 10-40 is an alkyl group, It is based on

[0170] Another preferred embodiment of Z represented by the formula (a2") for solid-liquid separation is R a and R b is a hydrogen atom; R 6 is a hydrogen atom; k is an integer from 1 to 3; k Q's are -O-; k R 7 Each independently has 1 to 3 linear C 10-40 A benzyl group with an alkyl group bonded via -O-, or 1 to 3 straight-chain C 10-40 is a cyclohexylmethyl group in which the alkyl group is bonded through -O-; and A ring B has k QR 7 In addition to the above, halogen atoms and C optionally substituted with halogen atoms 1-6 C optionally substituted with alkyl and halogen atoms 1-6 may have a substituent selected from the group consisting of alkoxy groups; It is based on

[0171] Another preferred embodiment of Z represented by the formula (a2") for solid-liquid separation is R a is a hydrogen atom; R b Formula (a3) ​​(wherein * is a bonding position, j is an integer of 0 to 3, j Q's are -O-, and j R 9 However, each independently, C 10-40 is an alkyl group, R 6 and R 8 are both hydrogen atoms. It is based on

[0172] Another preferred embodiment of Z represented by the formula (a2") for solid-liquid separation is R a is a hydrogen atom; R b Formula (a3) ​​(wherein * is a bonding position, j is an integer of 0 to 3, j Q's are -O-, and j R 9 However, each independently has a linear C 10-40 is an alkyl group, R8 is R 6 and together with the ring B and the ring C, form a condensed ring. It is based on

[0173] Another preferred embodiment of Z represented by the formula (a2") for solid-liquid separation is R a and R b together form an oxo group; R 6 is a hydrogen atom; k is an integer from 1 to 3; k Q's are -O-; and k R 7 However, each independently has a linear C 10-40 is an alkyl group, It is based on

[0174] Another preferred embodiment of Z represented by the formula (a2") for solid-liquid separation is R a and R b together form an oxo group; R 6 is a hydrogen atom; k is an integer from 1 to 3; k Q's are -O-; k R 7 Each independently has 1 to 3 linear C 10-40 A benzyl group with an alkyl group bonded via -O-, or 1 to 3 straight-chain C 10-40 is a cyclohexylmethyl group in which the alkyl group is bonded through -O-; and A ring B has k QR 7 In addition to the above, halogen atoms and C optionally substituted with halogen atoms 1-6 C optionally substituted with alkyl and halogen atoms 1-6 may have a substituent selected from the group consisting of alkoxy groups; It is based on

[0175] Pseudo-solid-phase protecting groups (gI) suitable for solid-liquid separation are preferably L is a succinyl group or a group represented by formula (a1′) (wherein R 1 and R 2 However, each independently, C 1-10 is an alkyl group, L 1 is a divalent phenylene group, and L 2 is a single bond; and YZ is 3,4,5-tris(octadecyloxy)benzyloxy group, 3,5-bis(docosyloxy)benzyloxy group, 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzyloxy group, 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzyloxy group, 3,4,5-tris(octadecyloxy)benzylamino group, 2,4-bis(docosyloxy)benzylamino group, 3,5-bis(docosyloxy)benzylamino group, bis(4-docosyloxyphenyl) methylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)benzyloxy]benzylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]benzylamino group, 2,4-bis(dodecyloxy)benzylamino group, phenyl(2,3,4-tris(octadecyloxy)phenyl)methylamino group, bis[4-(12-docosyloxydodecyloxy)phenyl]methylamino group, 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy] 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzylamino group, 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group, 3,5-bis(docosyloxy)cyclohexylmethyloxy group, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris(octadecyloxy)cyclohexylmethylamino group, 2,4-bis(docosyloxy)cyclohexylmethylamino group, 3,5-bis(docosyloxy)cyclohexylmethylamino group, bis(4-docosyloxycyclohexyl)methylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, 2,4-bis(dodecyloxy)cyclohexylmethylamino group, phenyl(2,3,4-tris(octadecyloxy)cyclohexyl)methylamino group, bis[4-(12-docosyloxydodecyloxy)cyclohexyl]methylamino group, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, or 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, or LY is a single bond or a succinyl-1,4-piperazinediyl group, and Z is a 3,4,5-tris(octadecyloxy)benzoyl group, a 3,5-bis(docosyloxy)benzoyl group, a 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzoyl group, or a 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzoyl group; It is based on

[0176] The pseudo-solid-phase protecting group (gI) suitable for solid-liquid separation is more preferably L is a succinyl group, and YZ is 3,4,5-tris(octadecyloxy)benzyloxy group, 3,5-bis(docosyloxy)benzyloxy group, 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzyloxy group, 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzyloxy group, 3,4,5-tris(octadecyloxy)benzylamino group, 2,4-bis(docosyloxy)benzylamino group, 3,5-bis(docosyloxy)benzylamino group, bis(4-docosyloxyphenyl) methylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)benzyloxy]benzylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]benzylamino group, 2,4-bis(dodecyloxy)benzylamino group, phenyl(2,3,4-tris(octadecyloxy)phenyl)methylamino group, bis[4-(12-docosyloxydodecyloxy)phenyl]methylamino group, 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy] 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzylamino group, 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group, 3,5-bis(docosyloxy)cyclohexylmethyloxy group, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris(octadecyloxy)cyclohexylmethylamino group, 2,4-bis(docosyloxy)cyclohexylmethylamino group, 3,5-bis(docosyloxy)cyclohexylmethylamino group, bis(4-docosyloxycyclohexyl)methylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, 2,4-bis(dodecyloxy)cyclohexylmethylamino group, phenyl(2,3,4-tris(octadecyloxy)cyclohexyl)methylamino group, bis[4-(12-docosyloxydodecyloxy)cyclohexyl]methylamino group, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, or 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, or LY is a single bond or a succinyl-1,4-piperazinediyl group, and Z is a 3,4,5-tris(octadecyloxy)benzoyl group, a 3,5-bis(docosyloxy)benzoyl group, a 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzoyl group, or a 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzoyl group.

[0177] The pseudo-solid-phase protecting group (gI) suitable for solid-liquid separation is further preferably L is a succinyl group, and YZ is 3,4,5-tris(octadecyloxy)benzyloxy group, 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group, 3,5-bis(docosyloxy)cyclohexylmethyloxy group, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris(octadecyloxy)cyclohexylmethylamino group, 2,4-bis(docosyloxy)cyclohexylmethylamino group, 3,5-bis(docosyloxy)cyclohexylmethylamino group, (i) a cyclohexylmethylamino group, a 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, a 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, a 2,4-bis(dodecyloxy)cyclohexylmethylamino group, a 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, or a 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group; or LY is a single bond or a succinyl-1,4-piperazinediyl group, and Z is a 3,4,5-tris(octadecyloxy)benzoyl group.

[0178] Pseudo-solid-phase protecting groups (gI) suitable for solid-liquid separation are particularly preferably L is a succinyl group, and YZ is a 3,4,5-tris(octadecyloxy)benzyloxy group, a 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group, or a phenyl(2,3,4-tris(octadecyloxy)phenyl)methylamino group; or LY is a succinyl-1,4-piperazinediyl group, and Z is a 3,4,5-tris(octadecyloxy)benzoyl group; It is based on

[0179] The pseudo-solid-phase protecting group (gI) suitable for solid-liquid separation is most preferably L is a succinyl group, and YZ is a 3,4,5-tris(octadecyloxy)benzyloxy group or a 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group; or LY is a succinyl-1,4-piperazinediyl group, and Z is a 3,4,5-tris(octadecyloxy)benzoyl group; It is based on

[0180] The pseudo-solid-phase protecting groups (gI) suitable for extraction are preferably the 2-{2,4-di(2',3'-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; the 3,5-di(2',3'-dihydrophytyloxy)benzyloxysuccinyl group; the 4-(2',3'-dihydrophytyloxy)benzyloxysuccinyl group; the 2-{1-[(2-chloro-5-(2',3'-dihydrophytyloxy)phenyl)]benzylaminocarbonyl}ethylcarbonyl group; the 3,4,5-tri(2',3'-dihydrophytyloxy) Benzyloxysuccinyl group;2-{3,4,5-tri(2',3'-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;2-{4-(2',3'-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;2-{2-[3',4',5'-tri(2",3"-dihydrophytyloxy)benzyloxy]-4-methoxybenzylaminocarbonyl}ethylcarbonyl group;2-{4-(2',3'-dihydrophytyloxy)-2-methoxybenzylaminocarbonyl}ethylcarbonyl group 4-(2',3'-dihydrophytyloxy)-2-methylbenzyloxysuccinyl group;2-{4-(2',3'-dihydrophytyloxy)-2-methylbenzylaminocarbonyl}ethylcarbonyl group;4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzyloxysuccinyl group;2-{4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylaminocarbonyl}ethylcarbonyl group;4-(3,7,11-trimethyldodecyloxy)benzyloxysuccinyl group 2-{4-(3,7,11-trimethyldodecyloxy)benzylaminocarbonyl}ethylcarbonyl group;2-{3,5-di(2',3'-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;2-{1-[2,3,4-tri(2',3'-dihydrophytyloxy)phenyl]benzylaminocarbonyl}ethylcarbonyl group;2-{1-[4-(2',3'-dihydrophytyloxy)phenyl]-4'-(2',3'-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;3,4,5-tris[3,4,5-tri(2',3'-dihydrophytyloxy)benzyl]benzyloxysuccinyl group; or 2-{3,4,5-tris[3,4,5-tri(2',3'-dihydrophytyloxy)benzyl]benzylaminocarbonyl}ethylcarbonyl group.

[0181] The "temporary protecting group removable under acidic conditions" that protects the hydroxyl group at the 5' position of nucleic acid b is not particularly limited as long as it can be deprotected under acidic conditions and can be used as a protecting group for a hydroxyl group. Examples of such a protecting group include trityl group, 9-(9-phenyl)xanthenyl group, 9-phenylthioxanthenyl group, bis(C1-6 alkoxy)trityl group such as 1,1-bis(4-methoxyphenyl)-1-phenylmethyl group (dimethoxytrityl group), and mono(C1-) such as 1-(4-methoxyphenyl)-1,1-diphenylmethyl group (monomethoxytrityl group). 18 Among these, from the viewpoints of ease of deprotection and availability, the temporary protecting group for the hydroxyl group is preferably a monomethoxytrityl group or a dimethoxytrityl group, and more preferably a dimethoxytrityl group.

[0182] L n1 The organic group in the above means a hydrocarbon group or a group in which a carbon atom in a hydrocarbon group is replaced with a heteroatom. Examples of heteroatoms include an oxygen atom, a nitrogen atom, and a sulfur atom. The organic group may have a substituent such as a hydroxyl group, an amino group, or an oxo group (=O). The hydroxyl group and the amino group that the organic group may have are preferably protected by a protective group. The shape of the organic group may be chain-like (straight-chain or branched-chain), cyclic, or a combination thereof.

[0183] The organic group may have a group that is functional to cells. The group that is functional to cells is preferably bonded to the end of the main chain or side chain of the organic group. Examples of the group that is functional to cells include "a group that improves the cell membrane permeability of a compound by improving the lipid solubility of the compound" and "a group that improves the uptake of a compound into cells via a cell membrane receptor". Examples of the "group that improves the cell membrane permeability of a compound by improving the lipid solubility of the compound" include cholesterol residues and tocopherol residues. Examples of the "group that improves the uptake of a compound into cells via a cell membrane receptor" include N-acetylgalactosamine residues. These groups that are functional to cells are described in International Publication No. WO 2017 / 104836.

[0184] -OL n1 Specific examples of -OH include the following (in the following formula, * indicates the bonding position with the phosphorus atom, and Ac indicates an acetyl group).

[0185] [ka]

[0186] L n1 is preferably C 2-6 It is preferably an alkylene group, more preferably an ethylene group.

[0187] The nucleic acid a, the nucleic acid α, and the nucleic acid b that are suitable for carrying out the production method of the present invention are described in detail below.

[0188] The nucleic acid a used in step (1) of the present invention includes, for example, a compound represented by the following formula (aI) (i.e., a nucleoside or oligonucleotide).

[0189] [ka]

[0190] [In the formula, m represents an integer equal to or greater than 0; m+1 Bases each independently represent an optionally protected nucleobase; m+1 X's each independently represent a hydrogen atom, a halogen atom, an optionally protected hydroxyl group, or a divalent organic group bonded to the 2-position carbon atom and the 4-position carbon atom; m+1 X's n1 each independently represents an oxygen atom or NH; m R 10 each independently represents an oxygen atom or a sulfur atom; m R p1 each independently represents a protecting group for a phosphate group; L, Y and Z are as defined above.]

[0191] Hereinafter, the compound represented by formula (aI) may be abbreviated as "compound (aI)". Compounds represented by other formulae may be abbreviated in the same manner.

[0192] The amino group of the nucleic acid base is preferably protected with a protecting group, such as an acetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a benzoyl group, an isobutyryl group, a (2-hexyl)decanoyl group, a dimethylformamidinyl group, and =NC(R 11 )-N(R 12 )(R 13 ) group (wherein, R 11 indicates a methyl group, and R 12 and R 13 are each independently C 1-5 Indicates an alkyl group or R 11 and R 12 may be taken together with the carbon atom and nitrogen atom to which they are attached to form a 5- or 6-membered nitrogen-containing hydrocarbon ring. 11 )-N(R 12 )(R 13An example of the 1-(dimethylamino)ethylidene group is a 1-(dimethylamino)ethylidene group. When compound (aI) has a plurality of amino groups, the protecting groups for the amino groups may be of one type or of two or more types.

[0193] When m is 0, compound (aI) is a nucleoside, and when m is 1 or more, compound (aI) is an oligonucleotide. m is preferably 49 or less, more preferably 29 or less, even more preferably 19 or less, particularly preferably 4 or less, and most preferably 2 or less.

[0194] As the halogen atom for X, a fluorine atom or a chlorine atom is preferable, and a fluorine atom is more preferable.

[0195] The protecting group for the optionally protected hydroxyl group of X is not particularly limited, and examples thereof include any protecting groups described in PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd Edition, published by JOHN WILLY & SONS (1999) and the like. Specifically, examples of the protecting group include a methyl group, a benzyl group, a p-methoxybenzyl group, a tert-butyl group, a methoxymethyl group, a methoxyethyl group, a 2-tetrahydropyranyl group, an ethoxyethyl group, a cyanoethyl group, a cyanoethoxymethyl group, a phenylcarbamoyl group, a 1,1-dioxothiomorpholine-4-thiocarbamoyl group, an acetyl group, a pivaloyl group, a benzoyl group, a trimethylsilyl group, a triethylsilyl group, a triisopropylsilyl group, a tert-butyldimethylsilyl group, a [(triisopropylsilyl)oxy]methyl (Tom) group, and a 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) group. The protecting group for the hydroxyl group, which may be protected, is preferably a triethylsilyl group, a triisopropylsilyl group, or a tert-butyldimethylsilyl group, and is particularly preferably a tert-butyldimethylsilyl group from the viewpoints of economy and availability.

[0196] The "divalent organic group bonded to the 2-position carbon atom and the 4-position carbon atom" of X is not particularly limited as long as it is bonded to the 2-position carbon atom and the 4-position carbon atom of the nucleoside. Examples of this divalent organic group include optionally substituted C 2-7 Alkylene groups, as well as -O- and -NR 33 -(R 33 is a hydrogen atom or C 1-6 (Indicating an alkyl group), -S-, -CO-, -COO-, -OCONR 34 -(R 34 is a hydrogen atom or C 1-6 (representing an alkyl group) and -CONR 35 -(R 35 is a hydrogen atom or C 1-6 a divalent linker selected from the group consisting of an alkyl group and an optionally substituted C 1-7 and a divalent organic group composed of an alkylene group. 1-7 Alkylene group and C 2-7 An example of the substituent that the alkylene group may have is a methylidene group (CH2=).

[0197] The "divalent organic group bonded to the 2-position carbon atom and the 4-position carbon atom" includes an optionally substituted C 2-7 Alkylene group, -OR i -(R i C is bonded to the 4th carbon atom 1-6 (representing an alkylene group), -O-NR 33 -R j -(R j C is bonded to the 4th carbon atom 1-6 R represents an alkylene group. 33 has the same meaning as above), -OR k -OR l -(R k is C 1-6 R represents an alkylene group. l C is bonded to the 4th carbon atom and bridged 1-6 alkylene group), and -OR i -(R i has the same meaning as above), -O-NR 33 -R j -(R j and R33 has the same meaning as above), -OR k -OR l -(R k and R l R is more preferably the same as defined above. i , R j , R k and R l C, as shown 1-6 The alkylene groups are each preferably independently a methylene group or an ethylene group.

[0198] Examples of the "divalent organic group bonded to the 2-position carbon atom and the 4-position carbon atom" include -O-CH2-, -O-CH2-CH2-, -O-NR 33 -CH2-(R 33 are as defined above), -O-CH2-O-CH2- is more preferred, and -O-CH2-, -O-CH2-CH2-, -O-NH-CH2-, -ON(CH3)-CH2-, and -O-CH2-O-CH2- (the left side is bonded to the 2-position carbon atom and the right side is bonded to the 4-position carbon atom in each case) are even more preferred.

[0199] Each of the (m+1) X's is independently preferably a hydrogen atom, a halogen atom, or an optionally protected hydroxyl group, more preferably a hydrogen atom or an optionally protected hydroxyl group.

[0200] R p1 The protecting group for the phosphate group is not particularly limited as long as it is removable under basic conditions and can be used as a protecting group for a phosphate group, but a group represented by -CH2CH2WG (WG represents an electron-withdrawing group) is preferred. Examples of the electron-withdrawing group of WG include a cyano group and a nitro group, and a cyano group is preferable.

[0201] m R p1 are preferably each independently a group represented by -CH2CH2WG. m+1 X's n1 is preferably an oxygen atom. The explanations of L, Y and Z are as above.

[0202] Compound (aI) is preferably a compound represented by the following formula (ai) (the definitions and explanations of the symbols in the following formula are as described above).

[0203] [ka]

[0204] Compound (aI) can be produced by a method known per se or a method equivalent thereto. For example, it can be produced according to the method described in WO 2017 / 104836.

[0205] Among the compounds (ai), the compounds represented by formula (a-II) (ie, nucleosides or oligonucleotides) are preferred.

[0206] [ka]

[0207] [In the formula, m, m Base, m+1 X, m R 10 , m R p1 , L, Y and Z are each independently as defined above; R 11 indicates a methyl group, and R 12 and R 13 are each independently C 1-5 Indicates an alkyl group or R 11 and R 12 may be joined together with the carbon atom and nitrogen atom to which they are attached to form a 5- or 6-membered nitrogen-containing hydrocarbon ring.

[0208] In formula (a-II), m R p1 are preferably each independently a group represented by -CH2CH2WG. In formula (a-II), m is preferably 0. That is, among compounds (a-II), compounds represented by formula (a-III) (i.e., nucleosides) are preferred (the definitions and explanations of the symbols in the following formula are as described above).

[0209] [ka]

[0210] In formula (a-II) and formula (a-III), R 11 is a methyl group, and R 12 and R 13 are each independently C 1-5 R is preferably an alkyl group. 11 , R 12 and R 13 However, it is more preferably a methyl group. The explanations of other symbols in formula (a-II) and formula (a-III) are as described above.

[0211] In the present invention, when an oligonucleotide having a phosphorothioated site in which the first residue has adenine as the nucleic acid base is produced, the use of compound (a-II) (particularly compound (a-III)) as the starting nucleoside, nucleotide or oligonucleotide can suppress the generation of branch bodies, etc. Here, the branch body refers to a by-product generated by the binding of the amino group to a monomer after the protective group of the amino group in the nucleic acid base of the target compound is removed.

[0212] Among the compounds (ai), the compounds represented by formula (a-IV) (ie, nucleosides or oligonucleotides) are preferred.

[0213] [ka]

[0214] [In the formula, m, m+1 Base, m+1 X, m R10 , m R p1 , L and Y are each independently as defined above; Z' is a member of the formula (a2"):

[0215] [ka]

[0216] [In the formula, * indicates bond position; R 6 is a hydrogen atom or R b is a group represented by the following formula (a3), R 8 may be taken together with a single bond or -O- to form a condensed ring with ring B and ring C; k represents an integer of 1 to 4; k Q's each independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH-, or -NHC(=O)-; k R 7 each independently represents a hydrocarbon group in which a linear aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linker, or represents an organic group having at least one aliphatic hydrocarbon group having one or more branched chains and having a total carbon number of 14 to 300; A ring B is a set of k QR 7 In addition to the above, halogen atoms and C optionally substituted with halogen atoms 1-6 C optionally substituted with alkyl and halogen atoms 1-6 may have a substituent selected from the group consisting of alkoxy groups; R a indicates a hydrogen atom; and R b is a hydrogen atom, or a group represented by the formula (a3):

[0217] [ka]

[0218] (In the formula, * indicates a bond position; j represents an integer of 0 to 4; j Q's each independently have the same meaning as defined above; j R 9 each independently represents a hydrocarbon group in which a linear aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linker, or represents an organic group having at least one aliphatic hydrocarbon group having one or more branched chains and having a total carbon number of 14 to 300; R 8 represents a hydrogen atom or R 6 may be taken together with a single bond or -O- to form a condensed ring with ring B and ring C; and A ring C has j QR 9 In addition to the above, halogen atoms and C optionally substituted with halogen atoms 1-6 C optionally substituted with alkyl and halogen atoms 1-6 ) which may have a substituent selected from the group consisting of alkoxy groups; R a and R b are taken together to form an oxo group.

[0219] In formula (a-IV), m R p1 are preferably each independently a group represented by -CH2CH2WG. In formula (a-IV), it is preferable that m is 0. The explanations of other symbols in formula (a-IV) are as described above. Among the compounds (a-IV), a compound represented by the following formula (aV) (i.e., a nucleoside) is preferred (the definitions and explanations of the symbols in the following formula are as described above).

[0220] [ka]

[0221] Among the compounds (aI), the compound (a-IV) (particularly, the compound (aV)) having Z' (i.e., a cyclohexylmethyl group structure) represented by the formula (a2") in the pseudo solid-phase protecting group has a higher solubility in a non-polar solvent than other compounds (aI) having Z (i.e., a benzyl group structure) represented by the formula (a2) in the pseudo solid-phase protecting group. Therefore, by using the compound (a-IV) (particularly, the compound (aV)), the production method of the present invention can be carried out at a higher concentration, and the productivity is greatly improved. The explanation of Z' represented by the formula (a2") is the same as the explanation of Z represented by the formula (a2") above.

[0222] In formulae (a-IV) and (aV), L is a succinyl group or a group represented by formula (a1′) (wherein R 1 and R 2 However, each independently, C 1-10 is an alkyl group, L 1 is a divalent phenylene group, and L 2 is a single bond; and Y-Z' is 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group, 3,5-bis(docosyloxy)cyclohexylmethyloxy group, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris(octadecyloxy)cyclohexylmethylamino group, 2,4-bis(docosyloxy)cyclohexylmethylamino group, 3,5-bis(docosyloxy)cyclohexylmethylami group a 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, a 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, a 2,4-bis(dodecyloxy)cyclohexylmethylamino group, a 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, or a 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group; The combination is preferred for solid-liquid separation.

[0223] In formulae (a-IV) and (aV), L is a succinyl group, and Y-Z' is 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group, 3,5-bis(docosyloxy)cyclohexylmethyloxy group, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris(octadecyloxy)cyclohexylmethylamino group, 2,4-bis(docosyloxy)cyclohexylmethylamino group, 3,5-bis(docosyloxy)cyclohexylmethylami group a 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, a 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, a 2,4-bis(dodecyloxy)cyclohexylmethylamino group, a 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, or a 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group; The combination is more preferred for solid-liquid separation.

[0224] In formulae (a-IV) and (aV), L is a succinyl group, and Y-Z' is a 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group, a 3,5-bis(docosyloxy)cyclohexylmethyloxy group, a 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, or a 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group; The combination is more preferred for solid-liquid separation.

[0225] In formulae (a-IV) and (aV), L is a succinyl group, and Y-Z' is a 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group; The combination is particularly preferred for solid-liquid separation.

[0226] Examples of the nucleic acid a include compounds represented by the following formula (a-VI) (ie, nucleosides or oligonucleotides).

[0227] [ka]

[0228] [In the formula, m+1 Bases 1 at least one of represents a nucleobase protected by -LYZ, and the rest represent optionally protected nucleobases; m X's n1 each independently represents an oxygen atom or NH; X n2 indicates a protected hydroxyl or amino group; m, m+1 Xs, m Rs 10 , m R p1 , L, Y and Z are each independently as defined above.]

[0229] In formula (a-VI), m+1 Base 1 At least one of is a nucleobase protected by -LYZ. The nucleobase and -LYZ are as described above. The optionally protected nucleobase is also as described above.

[0230] Protected hydroxyl group (X n2The protecting group for ) is not particularly limited, and may be, for example, any protecting group described in PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd Edition, John Wiley & Sons (1999), etc. Specific examples include a methyl group, a benzyl group, a p-methoxybenzyl group, a tert-butyl group, a methoxyethyl group, an ethoxyethyl group, a cyanoethyl group, a cyanoethoxymethyl group, a phenylcarbamoyl group, a 1,1-dioxothiomorpholine-4-thiocarbamoyl group, an acetyl group, a pivaloyl group, a benzoyl group, a triethylsilyl group, a triisopropylsilyl group, a tert-butyldimethylsilyl group, a [(triisopropylsilyl)oxy]methyl (Tom) group, a 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) group, etc. The protecting group of the hydroxyl group is preferably a triethylsilyl group, a triisopropylsilyl group, or a tert-butyldimethylsilyl group, and more preferably a tert-butyldimethylsilyl group from the viewpoints of economy and availability. The protection and deprotection of the hydroxyl group are well known, and can be carried out by, for example, the method described in Protective Groups in Organic Synthesis mentioned above.

[0231] Protected amino group (X n2The protecting group for ) is not particularly limited, and examples thereof include the protecting groups described in Greene's PROTECTIVE GROUPS in ORGANIC SYNTHESIS, 4th Edition, Wiley-Interscience Publishing (2006), etc. Specific examples of each protecting group include a pivaloyl group, a pivaloyloxymethyl group, an acetyl group, a trifluoroacetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a benzoyl group, an isobutyryl group, a (2-hexyl)decanoyl group, a dimethylformamidinyl group, a 1-(dimethylamino)ethylidene group, and a 9-fluorenylmethyloxycarbonyl group. The protecting group of the amino group is preferably an acetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a benzoyl group, an isobutyryl group, a (2-hexyl)decanoyl group, a dimethylformamidinyl group, or a 1-(dimethylamino)ethylidene group. Protection and deprotection of the amino group are well known, and can be carried out by the method described in the above-mentioned Protective Groups in Organic Synthesis.

[0232] m X's in formula (a-VI) n1 is preferably an oxygen atom. X in formula (a-VI) n2 is preferably a protected hydroxyl group. m R in formula (a-VI) p1 are preferably each independently a group represented by -CH2CH2WG. The explanations of other symbols in formula (a-VI) are as defined above. Compound (a-VI) is preferably a compound represented by the following formula (a-vi):

[0233] [ka]

[0234] [In the formula, R n4 represents a protecting group for a hydroxyl group; m, m+1 Bases 1 , m+1 X's, m R's 10 , m R p1 , L, Y and Z are each independently as defined above.]

[0235] R in formula (a-vi) n4 The explanation of X n2 This is the same as the explanation of the protecting group for the hydroxyl group in the above. The explanations of the other symbols in formula (a-vi) are as defined above.

[0236] Furthermore, examples of the nucleic acid α include compounds represented by the following formula (a-VII) (that is, nucleotides or oligonucleotides).

[0237] [ka]

[0238] [In the formula, m+1 X's n1 each independently represents an oxygen atom or NH; L n1 indicates an organic group; m, m+1 Base, m+1 X, m+1 R 10 , m+1 R p1 , L, Y and Z are each independently as defined above.]

[0239] L in formula (a-VII) n1 is preferably C 2-6 It is preferably an alkylene group, more preferably an ethylene group. m+1 X's in formula (a-VII) n1 is preferably an oxygen atom. m+1 R in formula (a-VII) p1 are preferably each independently a group represented by -CH2CH2WG. The explanation of other symbols in formula (a-VII) is as described above. Compound (a-VII) is preferably a compound represented by the following formula (a-vii) (the symbols in the following formula are defined and explained above).

[0240] [ka]

[0241] Compound (a-VII) can be produced by a method known per se or a method analogous thereto. n1 Compound (a-VII) in which is an oxygen atom and L is a succinyl group can be produced, for example, by the following steps. (i) condensing the compound ZYH with succinic anhydride to produce ZY-CO(CH)COOH; (ii) The obtained ZY-CO(CH2)2COOH is converted into a compound of the formula: HO-L in the presence of a condensing agent. n1 A compound represented by the formula: -OQ″ (wherein Q″ represents a temporary protecting group, L n1 represents an organic group.) and then deprotected to obtain ZY-CO(CH2)2CO-OL n1 Producing -OH, (iii) The obtained ZY-CO(CH2)2CO-OL n1 A phosphoramidite-modified nucleoside is reacted with the -OH to form a compound in which m is 0 and X n1 is an oxygen atom and L is a succinyl group, The above-mentioned condensation reaction and deprotection reaction are well known to those skilled in the art, and they can be carried out by those skilled in the art by appropriately setting the conditions.

[0242] Compound (a-VII) in which L is other than a succinyl group can also be produced by carrying out a similar reaction using a corresponding acid anhydride, a corresponding dicarboxylic acid halide, an active ester of a corresponding dicarboxylic acid, or the like, instead of succinic anhydride. n1Compound (a-VII) in which m is NH can be produced by carrying out a similar reaction using a nucleoside in which the 3'-amino group has been phosphoramidite-modified. Compound (a-VII) in which m is 1 or more can be produced by repeating the elongation process using compound (a-VII) in which m is 0 as the starting material.

[0243] The nucleic acid a or nucleic acid α used in step (1) of the present invention is preferably compound (aI), compound (a-VI) or compound (a-VII), more preferably compound (ai), compound (a-vi) or compound (a-vii), even more preferably compound (ai) or compound (a-vi), even more preferably compound (ai), even more preferably compound (a-II) or compound (a-IV), and particularly preferably compound (a-III) or compound (aV).

[0244] The nucleic acid b used in step (1) of the present invention has a 5'-position hydroxyl group protected with a protecting group (temporary protecting group). The protecting group for the hydroxyl group is not particularly limited as long as it can be deprotected under acidic conditions and can be used as a protecting group for the hydroxyl group. Examples of the protecting group include bis(C) groups such as trityl group, 9-(9-phenyl)xanthenyl group, 9-phenylthioxanthenyl group, and 1,1-bis(4-methoxyphenyl)-1-phenylmethyl group (dimethoxytrityl group). 1-6 alkoxy)trityl group, 1-(4-methoxyphenyl)-1,1-diphenylmethyl group (monomethoxytrityl group), etc. 1-18 Among these, from the viewpoints of ease of deprotection and availability, the temporary protecting group for the hydroxyl group is preferably a monomethoxytrityl group or a dimethoxytrityl group, and more preferably a dimethoxytrityl group.

[0245] The modification of the 3'-position hydroxyl group or 3'-position amino group in nucleic acid b by a method selected from the phosphoramidite method, the H-phosphonate method, the dihalophosphine method, and the oxazaphospholidine method can be carried out according to methods known in the art, as described in the explanation of step (1) above.

[0246] For an example of a method for producing nucleic acid b using the phosphoramidite method, the description of International Publication No. WO 2017 / 104836 can be referenced.

[0247] An example of the nucleic acid b is a compound represented by the following formula (bI) (i.e., a nucleoside or an oligonucleotide).

[0248] [ka]

[0249] [In the formula, q represents an integer equal to or greater than 0; q+1 Bases 2 each independently represents a nucleobase which may be protected with a protecting group selected from -LXZ and a protecting group used in nucleic acid synthesis; q+1 X's, q+1 R's p1 , q R 10 , L, X and Z are each independently as defined above; q+1 X's n1 each independently represents an oxygen atom or NH; Q" represents a temporary protecting group for a hydroxyl group that can be removed under acidic conditions; R 36 and R 37 each independently represents an alkyl group or a 5- or 6-membered saturated cyclic amino group formed together with the adjacent nitrogen atom, and such a saturated cyclic amino group may have one oxygen atom or one sulfur atom as a ring-constituting atom in addition to the nitrogen atom.

[0250] R 36 and R37 are each independently preferably C 1-10 It is an alkyl group or, together with the adjacent nitrogen atom, forms a 5- or 6-membered saturated cyclic amino group, more preferably C 1-10 is an alkyl group, more preferably C 1-6 It is an alkyl group.

[0251] The temporary protecting group for a hydroxyl group which can be removed under acidic conditions is as described above. Q″ is preferably a monomethoxytrityl group or a dimethoxytrityl group, more preferably a dimethoxytrityl group.

[0252] The amino group of the nucleic acid base in formula (bI) is preferably protected with a protecting group. Examples of the protecting group include -LXZ and a protecting group selected from protecting groups used in nucleic acid synthesis. The explanations of L, X, and Z are as above. Examples of the protecting group used in nucleic acid synthesis include an acetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a benzoyl group, an isobutyryl group, a (2-hexyl)decanoyl group, a dimethylformamidinyl group, and =NC(R 11 )-N(R 12 )(R 13 ) group (wherein, R 11 indicates a methyl group, and R 12 and R 13 are each independently C 1-5 Indicates an alkyl group or R 11 and R 12 may be taken together with the carbon atom and nitrogen atom to which they are attached to form a 5- or 6-membered nitrogen-containing hydrocarbon ring. 11 )-N(R 12 )(R 13 An example of the 1-(dimethylamino)ethylidene group is a 1-(dimethylamino)ethylidene group. When compound (bI) has a plurality of amino groups, the protecting groups for the amino groups may be of one type or of two or more types.

[0253] When q is 0, compound (bI) is a nucleoside, and when q is 1 or more, compound (bI) is an oligonucleotide. In compound (bI) used in this step, q is preferably 49 or less, more preferably 29 or less, even more preferably 19 or less, particularly preferably 4 or less, and most preferably 2 or less.

[0254] The q+1 Xs are each independently preferably a hydrogen atom, a halogen atom, or an optionally protected hydroxyl group, more preferably a hydrogen atom or an optionally protected hydroxyl group. Base 2 are each independently a nucleobase which may be protected with a protecting group preferably selected from protecting groups used in nucleic acid synthesis. q+1 R p1 are each independently preferably a group represented by CH2CH2WG. q+1 X's n1 is preferably an oxygen atom. The explanations of L, X and Z are as above. Compound (bI) is preferably a compound represented by the following formula (bi):

[0255] [ka]

[0256] [In the formula, q+1 Bases 3 each independently represents a nucleobase which may be protected with a protecting group used in nucleic acid synthesis, q, q+1 Xs, q+1 Rs p1 , q R 10 , Q”, R 36 and R 37 Each independently has the same meaning as defined above.

[0257] In formula (bi), a protecting group used in nucleic acid synthesis, q, q+1 X's, q+1 R's p1 , q R 10 , Q”, R 36and R 37 The explanation is as above.

[0258] The combination of nucleic acid a or nucleic acid α and nucleic acid b used in step (1) of the present invention is Preferred is compound (aI), compound (a-VI) or a combination of compound (a-VII) and compound (bI), More preferably, the compound (ai), the compound (a-vi) or the compound (a-vii) is combined with the compound (bi), Even more preferred is a combination of compound (ai) or compound (a-vi) with compound (bi), More preferred is a combination of compound (ai) and compound (bi), Even more preferred is a combination of compound (a-II) or compound (a-IV) with compound (bi), Particularly preferred is a combination of compound (a-III) or compound (aV) with compound (bi).

[0259] The method for producing an oligonucleotide having a phosphorothioate site of the present invention has been described in detail above. This method is a method for producing an oligonucleotide having a phosphorothioate site by elongating a nucleic acid chain in the "3' to 5' direction", which is currently common in the chain elongation reaction of a nucleic acid chain in the art. It is preferable that the nucleic acid A is a nucleoside, nucleotide, or oligonucleotide in which the 5'-position hydroxyl group is not protected and other groups may be protected by a protecting group used in nucleic acid synthesis, but the method for producing an oligonucleotide having a phosphorothioate site of the present invention can also be applied to the production of an oligonucleotide having a phosphorothioate site by elongating a nucleic acid chain in the opposite direction, "5' to 3'". The production of an oligonucleotide having a phosphorothioate site by elongating a nucleic acid chain in this "5' to 3' direction" can be performed by the method shown below.

[0260] [A] a step of condensing, in the presence of an antioxidant, a nucleoside, nucleotide or oligonucleotide (nucleic acid A-1) (preferably, the 3'-position in nucleic acid A-1 is a hydroxyl group) in which the 3'-position hydroxyl group or the 3'-position amino group is not protected and other groups may be protected with a protecting group used in nucleic acid synthesis or may be bound to a solid phase support, with a nucleoside, nucleotide or oligonucleotide (nucleic acid B-1) in which the 5'-position hydroxyl group has been modified by a method for forming a phosphite or phosphorous diester compound (e.g., a method selected from the phosphoramidite method, the H-phosphonate method, the dihalophosphine method and the oxazaphospholidine method) and other groups may be protected with a protecting group used in nucleic acid synthesis, to obtain a phosphite or phosphorous diester compound (step (1-1)); A method for producing an oligonucleotide having a phosphorothioate site comprising: [B] a step of condensing, in the presence of an antioxidant, a nucleoside, nucleotide or oligonucleotide (nucleic acid A-1) (preferably, the 3'-position in nucleic acid A-1 is a hydroxyl group) in which the 3'-position hydroxyl group or the 3'-position amino group is not protected and other groups may be protected with a protecting group used in nucleic acid synthesis or may be bound to a solid phase support, with a nucleoside, nucleotide or oligonucleotide (nucleic acid B-1) in which the 5'-position hydroxyl group has been modified by a method for forming a phosphite or phosphite diester (e.g., a method selected from the phosphoramidite method, the H-phosphonate method, the dihalophosphine method, and the oxazaphospholidine method) and other groups may be protected with a protecting group used in nucleic acid synthesis, to obtain a phosphite or phosphite diester, and further comprising: A method for producing an oligonucleotide having a phosphorothioate site, comprising the step of reacting the obtained phosphite or phosphorous diester with a sulfurizing agent (step (2-1)).

[0261] When a condensate is obtained by applying a method such as the phosphoramidite method, the H-phosphonate method, the dihalophosphine method, or the oxazaphosphoridine method using a nucleic acid A-1 having an amino group at the 3' position instead of a nucleic acid A-1 having a hydroxyl group at the 3' position, the bond between the 3' position and the phosphorus atom becomes an -NP bond instead of an -OP bond, but in this specification, for convenience, it is referred to as a "phosphite" or "phosphorous diester" as in the case of an -OP bond. Similarly, the corresponding "PO impurity" is referred to as a "phosphotriester" or "phosphodiester".

[0262] In the above-mentioned production method including the two steps, the definitions of each component such as "antioxidant", "sulfurizing agent", "nucleic acid A-1", and "nucleic acid B-1" and the detailed reaction conditions of the two steps are the same as those of each component and the reaction conditions of each step detailed in the above-mentioned production method of an oligonucleotide having a phosphorothioated site by elongation of a nucleic acid chain in the "3' to 5' direction". By referring to these, a person skilled in the art can appropriately produce an oligonucleotide having a phosphorothioated site by elongation of a nucleic acid chain in the "5' to 3' direction".

[0263] The present invention also includes a method for producing an oligonucleotide having a phosphorothioated site using a nucleoside, nucleotide or oligonucleotide (hereinafter sometimes referred to as "nucleic acid A") in which the 5'-position hydroxyl group is not protected and other groups may be bound to a solid-phase support used in nucleic acid synthesis, or a nucleoside, nucleotide or oligonucleotide (nucleic acid A-1) in which the 3'-position hydroxyl group or 3'-position amino group is not protected and other groups may be bound to a solid-phase support used in nucleic acid synthesis. When the production method of the present invention is carried out under solid-phase conditions, one group of nucleic acid A or nucleic acid A-1 is bound to a solid-phase support. The solid-phase support or resin used as the solid-phase support may be any support known in the art that is suitable for use in solid-phase synthesis. In this specification, the term "solid phase" includes that the nucleoside, nucleotide or oligonucleotide is bound or linked to the above-mentioned solid-phase support or resin via a conventional functional linker or handle group, and such linkers are also implied when the term "solid phase" is used in this context. Examples of solid phases are, for example, polystyrene supports (which may be further functionalized, for example, with p-methylbenzyl-hydrylamine) or rigid functionalized supports such as diatomaceous earth encapsulated polydimethylacrylamide (pepsin K), silica or microporous glass. The resin matrix of the solid phase may be composed of amphiphilic polystyrene-PEG resins or PEG-polyamide or PEG-polyester resins. Solid phase supports include, for example, Wang-PEG resin and Rink-amide PEG resin. EXAMPLES

[0264] The present invention will be described in more detail below with reference to examples, but these examples do not limit the scope of the present invention in any way. Furthermore, unless otherwise specified, the reagents, devices, and materials used in the present invention are commercially available. Furthermore, when amino acids and the like are represented by abbreviations in this specification, each representation is based on the abbreviations according to the IUPAC-IUB Commission on Biochemical Nomenclature or the commonly used abbreviations in the relevant field.

[0265] The meanings of the abbreviations used in the examples described below are as follows. SUC: Succinyl TOB: 3,4,5-tris(octadecyloxy)benzyloxy 2'-OMe-C-CE phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-cytidine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite DDTT: 5-[(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazole-3-thione ETT: 5-ethylthio-1H-tetrazole TFE: 2,2,2-trifluoroethanol TOF-MS: Time-of-flight mass spectrometer HO-CmT-SUC-TOB: (2'-O-methyl-cytidine 3'-[O-(2-cyanoethyl)] phosphorothionyl thymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) HO-CmCmT-SUC-TOB: (2'-O-methyl-cytidine 3'-[O-(2-cyanoethyl)]phosphorothionyl 2'-O-methyl-cytidine 3'-[O-(2-cyanoethyl)]phosphorothionyl thymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) HO-CmCmCmT-SUC-TOB: (2'-O-methyl-cytidine 3'-[O-(2-cyanoethyl)] phosphorothionyl 2'-O-methyl-cytidine 3'-[O-(2-cyanoethyl)] phosphorothionyl 2'-O-methyl-cytidine 3'-[O-(2-cyanoethyl)] phosphorothionyl thymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) HO-CmCmCmCmT-SUC-TOB: (2'-O-methyl-cytidine 3'-[O-(2-cyanoethyl)] phosphorothionyl (2'-O-methyl-cytidine 3'-[O-(2-cyanoethyl)] phosphorothionyl 2'-O-methyl-cytidine 3'-[O-(2-cyanoethyl)] phosphorothionyl 2'-O-methyl-cytidine 3'-[O-(2-cyanoethyl)] phosphorothionyl thymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) T-CE phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-thymidine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite HO-TT-SUC-TOB: (Thymidine 3'-[O-(2-cyanoethyl)] phosphorothionyl thymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) POS: 5-phenyl-3H-1,2,4-dithiazol-3-one

[0266] Piv-TOB: 3,4,5-tris(octadecyloxy)benzyl pivalate PADS: Phenylacetyl disulfide DMTr: 4,4'-dimethoxytrityl LC-TOF MS: Liquid chromatograph-time-of-flight mass spectrometer DBU: 1,8-diazabicyclo[5.4.0]-7-undecene PivCl: Pivaloyl chloride CMPT: N-(cyanomethyl)pyrrolidinium triflate

[0267] 2'-OMe-C-CE phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-N 4 -Benzoyl-2'-O-methyl-cytidine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite 2'-OMe-G-CE phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-N2 -Isobutyryl-2'-O-methyl-guanosine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite

[0268] 5-Me-dC(Bz)-CE phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-N 4 -Benzoyl-2'-deoxy-5-methylcytidine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite dG-CE phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-N 2 -Isobutyryl-deoxyguanosine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite dT-CE phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-deoxyglutamidine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite

[0269] 2'-O-TBDMS-C(Ac)-CE phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-N 4 -Acetyl-2'-O-(tert-butyldimethylsilyl)-cytidine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite

[0270] LNA-A(Bz)-CE phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-N 6 -Benzoyl-2'-O,4'-C-methylene-adenosine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite

[0271] 2'-FC(Bz)-CE phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-N 4 -Benzoyl-2'-fluoro-cytidine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite

[0272] dT-H-phosphonate TEA salt: 5'-O-(4,4'-dimethoxytrityl)-deoxythymidine-3'-H-phosphonate, triethylamine salt

[0273] HO-T-SUC-TOB: Deoxythymidin-3'-yl 3,4,5-tris(octadecyloxy)benzyl succinate HO-C-SUC-TOB: N 4 -Benzoyl-deoxycytidine-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate HO-TC-SUC-TOB: Deoxythymidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 4 -Benzoyl-deoxycytidine-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate

[0274] DMTrO-A·T·C·G·A·C·T·C·T·C·G·A·G·C·G·T·T·C·T·C-SUC-TOB (wherein "·" means a phosphorothionyl bond): 5'-O-(4,4'-dimethoxytrityl)-N 6 -[1-(Dimethylamino)ethylidene]-deoxyadenosine-3'-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 4 -Benzoyl-deoxycytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 2 -Isobutyryl-deoxyguanosine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 6 -[1-(Dimethylamino)ethylidene]-deoxyadenosine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 4 -Benzoyl-deoxycytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 4-Benzoyl-deoxycytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 4 -Benzoyl-deoxycytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 2 -Isobutyryl-deoxyguanosine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 6 -[1-(Dimethylamino)ethylidene]-deoxyadenosine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 2 -Isobutyryl-deoxyguanosine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 4 -Benzoyl-deoxycytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 2 -Isobutyryl-deoxyguanosine-3'-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine-3'-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 4 -Benzoyl-deoxycytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 4 -Benzoyl-deoxycytidine-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate

[0275] HO-CmT-SUC-TOB: N 4 -Benzoyl-2'-O-methyl-cytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate HO-GmCmT-SUC-TOB: N 2 -Isobutyryl-2'-O-methyl-guanosine 3'-[O-(2-cyanoethyl)]phosphoryl N 4-Benzoyl-2'-O-methyl-cytidine 3'-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate HO-2'-OMe-U-SUC-TOB: 2'-O-methyl-uridine-3'-yl 3,4,5-tris(octadecyloxy)benzyl succinate HO-Um-SUC-TOB: 2'-O-methyl-uridine-3'-yl 3,4,5-tris(octadecyloxy)benzyl succinate HO-CmUm-SUC-TOB: N 4 -Benzoyl-2'-O-methyl-cytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl 2'-O-methyl-uridine-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate HO-GUm-SUC-TOB: N 2 -Isobutyryl-deoxyguanosine 3'-[O-(2-cyanoethyl)]phosphorothionyl 2'-O-methyl-uridine-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate H.O.A. L T-SUC-TOB: N 6 -Benzoyl-2'-O,4'-C-methylene-adenosine-3'-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate HO-C f A L T-SUC-TOB: N 4 -Benzoyl-2'-fluoro-cytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 6 -Benzoyl-2'-O,4'-C-methylene-adenosine-3'-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate HO-rCC f A L T-SUC-TOB: N 4-Acetyl-2'-O-(tert-butyldimethylsilyl)-cytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 4 -Benzoyl-2'-fluoro-cytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 6 -Benzoyl-2'-O,4'-C-methylene-adenosine-3'-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate HO-(CH2)2-SUC-TOB: 3,4,5-Tris(octadecyloxy)benzyl 2-hydroxyethyl succinate H.O.A. L (CH2)2-SUC-TOB: N 6 -Benzoyl-2'-O,4'-C-methylene-adenosine-3'-[O-(2-cyanoethyl)]phosphorothionyl ethyloxy-[3,4,5-tris(octadecyloxy)benzyl]succinate HO-C f A L (CH2)2-SUC-TOB: N 4 -Benzoyl-2'-fluoro-cytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 6 -Benzoyl-2'-O,4'-C-methylene-adenosine-3'-[O-(2-cyanoethyl)]phosphorothionyl ethyloxy-[3,4,5-tris(octadecyloxy)benzyl]succinate HO-rCC f A L (CH2)2-SUC-TOB: N 4 -Acetyl-2'-O-(tert-butyldimethylsilyl)-cytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 4 -Benzoyl-2'-fluoro-cytidine-3'-[O-(2-cyanoethyl)]phosphorothionyl N 6 -Benzoyl-2'-O,4'-C-methylene-adenosine-3'-[O-(2-cyanoethyl)]phosphorothionyl ethyloxy-[3,4,5-tris(octadecyloxy)benzyl]succinate H.O.T. H T-SUC-TOB: Deoxythymidine-3'-phosphonate Deoxythymidine-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate H.O.T. OH T-SUC-TOB: Deoxythymidine-3'-phosphorothionyl deoxythymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate HO-GmT-SUC-TOB: N 2 -Isobutyryl-2'-O-methyl-guanosine 3'-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate H.O.T. SCNEt T-SUC-TOB: Deoxythymidine-3'-[S-(2-cyanoethyl)]phosphorothionyl deoxythymidine-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate

[0276] The TOB adducts described below were synthesized by the method described in International Publication No. 2012 / 157723. Phosphoramidite monomers such as 2'-OMe-C-CE phosphoramidite used in the examples and comparative examples were commercially available products.

[0277] Example 1: Synthesis of phosphorothioated dimers to pentamers (with antioxidant) Under an argon atmosphere, HO-T-SUC-TOB (248.2 mg, 0.20 mmol) was placed in a 50 mL two-neck flask, and dehydrated dichloromethane (10.0 mL) and dehydrated acetonitrile (1.0 mL) were added to dissolve the TOB. 2'-OMe-C-CE phosphoramidite (518.5 mg, 0.60 mmol) and methyldiphenylphosphine (37.8 μL, 0.20 mmol) were then added and stirred for 30 minutes. Then, ETT (5-ethylthio-1H-tetrazole, 78.1 mg, 0.60 mmol) was added and stirred at room temperature for 1 hour. Next, TFE (2,2,2-trifluoroethanol, 219 μL, 3.01 mmol) was added to the reaction solution and stirred at room temperature for 30 minutes, after which 2,6-xylidine (638 μL, 5.18 mmol) and DDTT (5-[(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazole-3-thione, 215.6 mg, 1.05 mmol) were added in sequence and stirred at room temperature for 1 hour. Analysis of the resulting dimer (sometimes referred to as 2mer; the same applies below) showed that the PO impurity was 0.09%. m / z(TOF-MS):Calcd.1729.07, Found 1730.08 [M+H] + The HO-CmCmT-SUC-TOB trimer was synthesized (290.8 mg) using the HO-CmT-SUC-TOB dimer (279.6 mg, 0.16 mmol) and 2'-OMe-C-CE phosphoramidite (419.2 mg, 0.485 mmol) in the same manner as above. Analysis of the resulting trimer revealed that the PO impurity was 0.25%. m / z(TOF-MS):Calcd.2221.16, Found 2222.17 [M+H] + The tetramer HO-CmCmCmT-SUC-TOB was synthesized in the same manner using the extended trimer HO-CmCmT-SUC-TOB (290.8 mg, 0.13 mmol) and 2'-OMe-C-CE phosphoramidite (340.3 mg, 0.394 mmol). Analysis of the resulting tetramer revealed that it contained 0.27% PO impurity. m / z(TOF-MS):Calcd.2713.24, Found 2714.23 [M+H] + Similarly, the pentamer HO-CmCmCmCmT-SUC-TOB was synthesized (254 mg) using the extended tetramer HO-CmCmCmT-SUC-TOB (283.0 mg, 0.10 mmol) and 2'-OMe-C-CE phosphoramidite (270.1 mg, 0.313 mmol). Analysis of the resulting pentamer revealed that it contained 0.30% PO impurity. m / z(TOF-MS):Calcd.3205.33, Found 1604.69 [M+2H] 2+ Table 1 shows the content of PO impurities for each chain length when an antioxidant is used.

[0278] [Table 1]

[0279] Comparative Example 1: Synthesis of phosphorothioated dimers to pentamers (without antioxidant) Under an argon atmosphere, HO-T-SUC-TOB (247.6 mg, 0.20 mmol) was placed in a 50 mL two-neck flask, and dehydrated dichloromethane (10.0 mL) and dehydrated acetonitrile (1.0 mL) were added to dissolve it. Then, 2'-OMe-C-CE phosphoramidite (518.4 mg, 0.60 mmol) and ETT (5-ethylthio-1H-tetrazole, 78.1 mg, 0.60 mmol) were added in that order, and the mixture was stirred at room temperature for 1 hour. Next, TFE (2,2,2-trifluoroethanol, 218.6 μL, 3.0 mmol) was added to the reaction solution and stirred at room temperature for 30 minutes, after which 2,6-xylidine (380.6 μL, 3.0 mmol) and DDTT (5-[(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazole-3-thione, 129.3 mg, 0.63 mmol) were added in sequence and stirred at room temperature for 1 hour. Analysis of the resulting dimer showed that the PO impurity was 0.11%. m / z(TOF-MS):Calcd.1729.07, Found 1730.08 [M+H] + The trimer HO-CmCmT-SUC-TOB was synthesized (349.1 mg) using the dimer HO-CmT-SUC-TOB (297.4 mg, 0.17 mmol) and 2'-OMe-C-CE phosphoramidite (445.5 mg, 0.52 mmol) in the same manner as above. Analysis of the resulting trimer revealed that the PO impurity was 0.46%. m / z(TOF-MS):Calcd.2221.16, Found 2222.17 [M+H]+ Similarly, the tetramer HO-CmCmCmT-SUC-TOB was synthesized (415.3 mg) using the trimer HO-CmCmT-SUC-TOB (349.1 mg, 0.16 mmol) and 2'-OMe-C-CE phosphoramidite (407.5 mg, 0.47 mmol). Analysis of the resulting tetramer revealed that the PO impurity was 0.68%. m / z(TOF-MS):Calcd.2713.24, Found 2714.23 [M+H] + Similarly, the pentamer HO-CmCmCmCmT-SUC-TOB was synthesized (439.3 mg) using the tetramer HO-CmCmCmT-SUC-TOB (415.3 mg, 0.15 mmol) and 2'-OMe-C-CE phosphoramidite (399.2 mg, 0.46 mmol). Analysis of the resulting pentamer revealed that the PO impurity was 0.99%. m / z(TOF-MS):Calcd.3205.33, Found 1604.69 [M+2H] 2+ Table 2 shows the content of PO impurities for each chain length when no antioxidant was used.

[0280] [Table 2]

[0281] Example 2: Synthesis of trimers from phosphorothioated dimers (in the presence of various antioxidants) Under an argon atmosphere, HO-TT-SUC-TOB (56.4mg, 0.035mmol) synthesized in the same manner as in Example 1 was placed in a 20mL two-neck flask, using T-CE phosphoramidite instead of 2'-OMe-C-CE phosphoramidite in Example 1, and dissolved in dehydrated dichloromethane (1.8mL) and dehydrated acetonitrile (0.5mL). Then, 2'-OMe-C-CE phosphoramidite (91.8mg, 0.105mmol) and various antioxidants (0.035mmol) were added and stirred for 30 minutes. Then, ETT (5-ethylthio-1H-tetrazole, 14mg, 0.105mmol) was added and stirred at room temperature for 3 hours. Next, TFE (2,2,2-trifluoroethanol, 38.4 μL, 0.53 mmol) was added to the reaction solution and stirred at room temperature for 2 hours, after which 2,6-xylidine (50.2 μL, 0.407 mmol) and POS (5-phenyl-3H-1,2,4-dithiazol-3-one, 33.3 mg, 0.171 mmol) were added in sequence and stirred at room temperature for 1 hour, after which each reaction solution was analyzed. The results regarding the content ratio of PO impurities are shown in Table 3.

[0282] Comparative Example 2: Synthesis of trimer from phosphorothioated dimer (without antioxidant) The same procedure as in Example 2 was repeated except that the various antioxidants (0.035 mmol) of Example 2 were not used. The results regarding the content ratio of PO impurities are shown in Table 3.

[0283] [Table 3]

[0284] The following are examples of the synthesis of various phosphorothioated oligomers in the presence of an antioxidant.

[0285] Example 3: Synthesis of phosphorothioated tetramer (phosphoramidite method) Extension reaction Under an argon atmosphere, HO-T-SUC-TOB (300 mg, 242 μmol) and Piv-TOB (450 mg, 451 μmol) were placed in a 200 mL two-neck flask, and dehydrated dichloromethane (12.1 mL) and dehydrated acetonitrile (3.6 mL) were added to dissolve them. Then, triphenylphosphine (31.8 mg, 121 μmol), LNA-A(Bz)-CE phosphoramidite (644 mg, 727 μmol), and 5-ethylthio-1H-tetrazole (94.6 mg, 727 μmol) were added in that order, and the mixture was stirred at room temperature for 1.0 hour. Next, 2,2,2-trifluoroethanol (265 μL, 3.6 mmol) was added to the reaction solution and stirred at room temperature for 30 minutes, after which 2,6-xylidine (506 μL, 4.1 mmol) and DDTT (174 mg, 848 μmol) were added in that order and stirred at room temperature for 1.7 hours. Then, indole (424 mg, 3.6 mmol) and trifluoroacetic acid (1.1 mL, 14.4 mmol) were added in that order and stirred at room temperature for 1.0 hour. Next, pyridine (3.5 mL, 43.0 mmol) and water (180 μL) were added in that order and stirred at room temperature for 1.0 hour. Then, acetonitrile (112 mL) was added, and the precipitated solid was suction filtered using a Kiriyama funnel, dried, and the dimer HO-A was obtained. L T-SUC-TOB was obtained as a white solid (829.9 mg, 95%). m / z:Calcd.1751.07, Found 1753.09[M+H] + In the same manner as above, the white solid (830 mg) obtained above was used with 2'-FC(Bz)-CE phosphoramidite and 4,5-dicyanoimidazole instead of 5-ethylthio-1H-tetrazole to prepare the trimer HO-C. f A L T-SUC-TOB was obtained as a white solid (780.1 mg, 83%). m / z:calcd.2231.13, found 1117.07[M+2H] 2+ Similarly, the tetramer HO-rCC was prepared by using the white solid (718 mg) obtained above, 2'-O-TBDMS-C(Ac)-CE phosphoramidite, and ethylthio-1H-tetrazole instead of 4,5-dicyanoimidazole 5-. f A L T-SUC-TOB was obtained as a white solid (758.5 ​​mg, 94%). m / z:calcd.2761.27, found 1382.15 [M+2H] 2+ Deprotection reaction The white solid (10 mg) obtained above and 28% aqueous ammonia (5 mL) were placed in an autoclave and heated at 65°C for 4 hours, then cooled to room temperature. Insoluble matter in the reaction solution was removed using a syringe filter, and the solution was concentrated under reduced pressure using a centrifugal evaporator. The concentrated solution was then freeze-dried to obtain the target product, 2'-O-(tert-butyldimethylsilyl)-cytidine-3'-phosphorothionyl-2'-fluoro-cytidine-3'-phosphorothionyl-2'-O,4'-C-methylene-adenosine-3'-phosphorothionyl-deoxythymidine. m / z:calcd.1357.24,found 1356.23[MH] -

[0286] Example 4: Synthesis of phosphorothioated dimers (phosphoramidite method) Under an argon atmosphere, HO-C-SUC-TOB (3.0 g, 2.3 mmol) and Piv-TOB (3.0 g, 3 mmol) were placed in a 1000 mL three-neck flask, and dehydrated chloroform (113 mL) and dehydrated acetonitrile (34 mL) were added to dissolve them. Then, triphenylphosphine (296 mg, 1.1 mmol), dT-CE phosphoramidite (3.7 g, 5.0 mmol), and 5-ethylthio-1H-tetrazole (648 mg, 5.0 mmol) were added in that order, and the mixture was stirred at room temperature for 1.5 hours. Next, 2,2,2-trifluoroethanol (1.8 mL, 24.9 mmol) was added to the reaction solution and stirred at room temperature for 30 minutes, after which 2,6-xylidine (2.2 mL, 17.6 mmol) and POS (1.2 g, 6.1 mmol) were added successively and stirred at room temperature for 1.5 hours. Then, 2,3-dimethylfuran (2.6 mL, 24.8 mmol) and trifluoroacetic acid (5.6 mL, 72.8 mmol) were added in that order, and the mixture was stirred at room temperature for 1.5 hours. Next, pyridine (44.1 mL, 546 mmol) and water (960 μL) were added in that order to the reaction solution, and the mixture was stirred at room temperature for 1.5 hours. Then, acetonitrile (1 L) was added, and the precipitated solid was suction filtered using a Kiriyama funnel and then dried to obtain the dimer HO-TC-SUC-TOB as a white solid. (6.7 g, 99%)

[0287] Example 5: Synthesis of phosphorothioated trimers (phosphoramidite method) Extension reaction The same procedure as in Example 3 was repeated except that HO-(CH2)2-SUC-TOB (256 mg) was used instead of HO-T-SUC-TOB to prepare HO-A L (CH2)2-SUC-TOB, followed by dimer HO-C f A L (CH2)2-SUC-TOB, and finally the trimer HO-rCC f A L (CH2)2-SUC-TOB was obtained as a white solid (691mg). m / z:calcd.2581.22, found 1292.11 [M+2H] 2+ Deprotection reaction The white solid (10 mg) obtained above and 28% aqueous ammonia (5 mL) were placed in an autoclave and heated at 65°C for 4 hours, then cooled to room temperature. Insoluble matter in the reaction solution was removed using a syringe filter, and the solution was concentrated under reduced pressure using a centrifugal evaporator. The concentrated solution was then freeze-dried to obtain the target product, 2-[2'-O-(tert-butyldimethylsilyl)-cytidine-3'-phosphorothionyl-2'-fluoro-cytidine-3'-phosphorothionyl-2'-O,4'-C-methylene-adenosine-3'-phosphorothionyl]ethanol. m / z:calcd.1177.18,found 1176.18[MH] -

[0288] Example 6: Synthesis of phosphorothioated 20-mer (phosphoramidite method) Extension reaction Using dichloromethane instead of chloroform, the same procedure as in Example 4 was repeated 18 times to obtain the 20-mer DMTrO-A·T·C·G·A·C·T·C·T·C·G·A·G·C·G·T·T·C·T·C-SUC-TOB (where "·" represents a phosphorothionyl bond) as a white solid (8.5 g). Deprotection reaction The obtained white solid (5 mg) and 28% aqueous ammonia (5 mL) were placed in an autoclave and heated at 65°C for 4 hours, and then cooled to room temperature. Insoluble matter in the reaction solution was removed using a syringe filter, and the solution was concentrated under reduced pressure using a centrifugal evaporator. The concentrated solution was then freeze-dried to obtain the target product, 5'-O-(4,4'-dimethoxytrityl)-deoxyadenosine-3'-phosphorothionyl-deoxythymidine-3'-phosphorothionyl-deoxycytidine-3'-phosphorothionyl-deoxyguanosine-3'-phosphorothionyl-deoxyadenosine-3'-phosphorothionyl-deoxycytidine-3'-phosphorothionyl-deoxythymidine-3'-phosphorothionyl-deoxycytidine-3'-phosphorothionyl-deoxythymidine-3'-phosphorothionyl The resulting mixture was 3'-phosphorothionyl-deoxycytidine-3'-phosphorothionyl-deoxyguanosine-3'-phosphorothionyl-deoxyadenosine-3'-phosphorothionyl-deoxyguanosine-3'-phosphorothionyl-deoxycytidine-3'-phosphorothionyl-deoxyguanosine-3'-phosphorothionyl-deoxythymidine-3'-phosphorothionyl-deoxythymidine-3'-phosphorothionyl-deoxycytidine-3'-phosphorothionyl-deoxythymidine-3'-phosphorothionyl-deoxycytidine-3'-phosphorothionyl-deoxythymidine-3'-phosphorothionyl-deoxycytidine. m / z:calcd.6648.73,found 1661.67[M-4H] 4ー

[0289] Example 7: Synthesis of phosphorothioated dimers (H-phosphonate method) Under an argon atmosphere, HO-T-SUC-TOB (100 mg, 80.8 μmol), dT-H-phosphonate TEA salt (116.0 mg, 163 μmol), and triphenylphosphine (10.0 mg, 38.1 μmol) were placed in a 50 mL two-neck flask, and dissolved in dehydrated dichloromethane (2.0 mL) and dehydrated pyridine (2.0 mL). Then, PivCl (29.5 μl, 242 μmol) was added and stirred at room temperature for 1.0 hour. Next, DBU (48.4 μL, 324 μmol) and DDTT (34.3 mg, 167 μmol) were added to the reaction solution and stirred at room temperature for 30 minutes. Then, acetonitrile (30 mL) was added, and the precipitated solid was suction filtered with a Kiriyama funnel and then dried to obtain a white solid (145.7 mg). The obtained solid was placed in a 50 mL two-neck flask under an argon atmosphere, and dehydrated dichloromethane (4.0 mL) and dehydrated acetonitrile (1.2 mL) were added to dissolve it. Then, indole (46.3 mg, 395 μmol) and trifluoroacetic acid (72.6 μL, 948 μmol) were added in that order, and the mixture was stirred at room temperature for 1.0 hour. Next, pyridine (220 μL, 2.7 mmol) and water (42 μL) were added in that order to the reaction solution, and the mixture was stirred at room temperature for 1.0 hour. Then, acetonitrile (30 mL) was added, and the precipitated solid was suction filtered using a Kiriyama funnel, dried, and dimer HO-T OH T-SUC-TOB was obtained as a white solid (85.9 mg, 71%). m / z:calcd.1557.01, found 1558.02 [M+H] +

[0290] Example 8: Synthesis of phosphorothioated dimers (H-phosphonate method) Under an argon atmosphere, HO-T-SUC-TOB (101 mg, 81.3 μmol), dT-H-phosphonate TEA salt (115.4 mg, 163 μmol), and triphenylphosphine (10.8 mg, 41.1 μmol) were placed in a 50 mL two-neck flask, and dehydrated dichloromethane (2.0 mL) and dehydrated pyridine (2.0 mL) were added to dissolve. PivCl (29.5 μl, 242 μmol) was then added and stirred at room temperature for 1.0 hour. Acetonitrile (30 mL) was then added, and the precipitated solid was suction filtered with a Kiriyama funnel to obtain a white solid (145.2 mg). The obtained solid was placed in a 50 mL two-neck flask under an argon atmosphere, and dehydrated dichloromethane (4.0 mL) and dehydrated acetonitrile (1.2 mL) were added to dissolve. Then, indole (47.2 mg, 403 μmol) and trifluoroacetic acid (73.0 μL, 953 μmol) were added in that order, and the mixture was stirred at room temperature for 1.0 hour. Next, pyridine (230 μL, 2.8 mmol) and water (42 μL) were added in that order to the reaction solution, and the mixture was stirred at room temperature for 1.0 hour. Then, acetonitrile (30 mL) was added, and the precipitated solid was suction filtered using a Kiriyama funnel, dried, and the dimer HO-T was obtained. H T-SUC-TOB was obtained as a white solid (104 mg, 86%). m / z:calcd.1525.03, found 1526.05[M+H] +

[0291] Example 9: Synthesis of phosphorothioated dimer (sulfurizing agent: PADS) Under an argon atmosphere, HO-T-SUC-TOB (98.8 mg, 79.6 μmol) and Piv-TOB (102 mg, 103 μmol) were placed in a 50 mL two-neck flask, and dehydrated dichloromethane (4.0 mL) and dehydrated acetonitrile (1.2 mL) were added to dissolve them. Then, triphenylphosphine (13.2 mg, 50.3 μmol), 2'-OMe-G-CE phosphoramidite (144 mg, 165 μmol), and 5-ethylthio-1H-tetrazole (21.2 mg, 163 μmol) were added in that order, and the mixture was stirred at room temperature for 1.0 hour. Next, 2,2,2-trifluoroethanol (58.9 μL, 810 μmol) was added to the reaction solution and stirred at room temperature for 30 minutes, then 2,6-xylidine (71.2 μL, 577 μmol) and bisphenylacetyl disulfide (62.9 mg, 208 μmol) were added in sequence and stirred at room temperature for 1.0 hour, then indole (98.2 mg, 838 μmol) and trifluoroacetic acid (220 μL, 2.87 mmol) were added in sequence and stirred at room temperature for 1.0 hour. Next, pyridine (690 μL, 8.6 mmol) and methanol (57 μL) were added to the reaction solution in that order, and the mixture was stirred at room temperature for 1.0 hour. Then, acetonitrile (37 mL) was added, and the precipitated solid was suction filtered using a Kiriyama funnel, and then dried to obtain the dimer HO-GmT-SUC-TOB as a white solid (222.7 mg, 93%). m / z(TOF-MS):Calcd.1735.09, Found 1737.11[M+H] +

[0292] Example 10: Synthesis of phosphorothioated dimer (sulfurizing agent: xanthan hydride) Under an argon atmosphere, HO-T-SUC-TOB (96.8 mg, 78.2 μmol) and Piv-TOB (99.3 mg, 99.5 μmol) were placed in a 50 mL two-neck flask, and dehydrated dichloromethane (4.0 mL) and dehydrated acetonitrile (1.2 mL) were added to dissolve them. Then, triphenylphosphine (11.5 mg, 43.8 μmol), 2'-OMe-G-CE phosphoramidite (140 mg, 161 μmol), and 5-ethylthio-1H-tetrazole (20.9 mg, 161 μmol) were added in that order, and the mixture was stirred at room temperature for 1.0 hour. Next, 2,2,2-trifluoroethanol (58.9 μL, 810 μmol) was added to the reaction solution and stirred at room temperature for 30 minutes, after which 2,6-xylidine (71.2 μL, 577 μmol) and xanthan hydride (31.2 mg, 208 μmol) were added successively and stirred at room temperature for 1.0 hour. Then, indole (96.5 mg, 824 μmol) and trifluoroacetic acid (220 μL, 2.87 mmol) were added in that order, and the mixture was stirred at room temperature for 1.0 hour. Next, pyridine (690 μL, 8.6 mmol) and methanol (57 μL) were added in that order to the reaction solution, and the mixture was stirred at room temperature for 1.0 hour. Then, acetonitrile (37 mL) was added, and the precipitated solid was suction filtered using a Kiriyama funnel, and then dried to obtain the dimer HO-GmT-SUC-TOB as a white solid. (217.9 mg, 91%) m / z(TOF-MS):Calcd.1735.09, Found 1737.11[M+H] +

[0293] Example 11: Synthesis of phosphorothioated dimer (sulfurizing agent: sulfur) Under an argon atmosphere, HO-T-SUC-TOB (101 mg, 81.3 μmol) and Piv-TOB (98.7 mg, 98.9 μmol) were placed in a 50 mL two-neck flask, and dehydrated dichloromethane (4.0 mL) and dehydrated acetonitrile (1.2 mL) were added to dissolve them. Then, triphenylphosphine (12.0 mg, 45.8 μmol), 2'-OMe-G-CE phosphoramidite (141 mg, 162 μmol), and 5-ethylthio-1H-tetrazole (21.9 mg, 168 μmol) were added in that order, and the mixture was stirred at room temperature for 1.0 hour. Next, 2,2,2-trifluoroethanol (58.9 μL, 810 μmol) was added to the reaction solution and stirred at room temperature for 30 minutes, after which 2,6-xylidine (71.2 μL, 577 μmol) and sulfur (13.0 mg, 405 μmol) were added successively and stirred at room temperature for 1.0 hour. Then, indole (96.0 mg, 819 μmol) and trifluoroacetic acid (220 μL, 2.87 mmol) were added in that order, and the mixture was stirred at room temperature for 1.0 hour. Next, pyridine (690 μL, 8.6 mmol) and methanol (57 μL) were added in that order to the reaction solution, and the mixture was stirred at room temperature for 1.0 hour. Then, acetonitrile (37 mL) was added, and the precipitated solid was suction filtered using a Kiriyama funnel, and then dried to obtain the dimer HO-GmT-SUC-TOB as a white solid. (221.2 mg, 92%) m / z(TOF-MS):Calcd.1735.09, Found 1737.11[M+H] +

[0294] Example 12: Synthesis of phosphorothioated dimer (deprotection: trichloroacetic acid) Under an argon atmosphere, HO-Um-SUC-TOB (102.4 mg, 81.7 μmol) and Piv-TOB (105 mg, 105 μmol) were placed in a 50 mL two-neck flask, and dehydrated dichloromethane (4.0 mL) and dehydrated acetonitrile (1.2 mL) were added to dissolve them. Then, triphenylphosphine (9.8 mg, 37.3 μmol), dG-CE phosphoramidite (135.1 mg, 161 μmol), and 5-ethylthio-1H-tetrazole (21.4 mg, 164 μmol) were added in that order, and the mixture was stirred at room temperature for 1.0 hour. Next, 2,2,2-trifluoroethanol (58.1 μL, 797 μmol) was added to the reaction solution and stirred at room temperature for 30 minutes, after which 2,6-xylidine (118 μL, 955 μmol) and DDTT (40.9 mg, 199 μmol) were added successively and stirred at room temperature for 1.0 hour. Then, indole (93.6 mg, 799 μmol) and trichloroacetic acid (698 mg, 4.27 mmol) were added in that order, and the mixture was stirred at room temperature for 1.0 hour. Next, pyridine (1.01 mL, 12.6 mmol) and water (66 μL) were added in that order to the reaction solution, and the mixture was stirred at room temperature for 1.0 hour. Then, acetonitrile (42 mL) was added, and the precipitated solid was suction filtered using a Kiriyama funnel, and then dried to obtain the dimer HO-GUm-SUC-TOB as a white solid. (241.5 mg) m / z(TOF-MS):calcd.1721.07, found 1723.10[M+H] +

[0295] Example 13: Synthesis of phosphorothioated dimer (deprotection: dichloroacetic acid) Under an argon atmosphere, HO-Um-SUC-TOB (99.6 mg, 79.4 μmol) and Piv-TOB (97.9 mg, 98.0 μmol) were placed in a 50 mL two-neck flask, and dehydrated dichloromethane (4.0 mL) and dehydrated acetonitrile (1.2 mL) were added to dissolve them. Then, triphenylphosphine (10.8 mg, 41.1 μmol), dG-CE phosphoramidite (134.2 mg, 160 μmol), and 5-ethylthio-1H-tetrazole (20.9 mg, 161 μmol) were added in that order, and the mixture was stirred at room temperature for 1.0 hour. Next, 2,2,2-trifluoroethanol (58.1 μL, 797 μmol) was added to the reaction solution and stirred at room temperature for 30 minutes, after which 2,6-xylidine (118 μL, 955 μmol) and DDTT (40.9 mg, 199 μmol) were added successively and stirred at room temperature for 1.0 hour. Then, indole (93.2 mg, 796 μmol) and dichloroacetic acid (478 μl, 5.78 mmol) were added in that order, and the mixture was stirred at room temperature for 1.0 hour. Next, pyridine (1.40 mL, 17.4 mmol) and water (61 μL) were added in that order to the reaction solution, and the mixture was stirred at room temperature for 1.0 hour. Then, acetonitrile (40 mL) was added, and the precipitated solid was suction filtered using a Kiriyama funnel, and then dried to obtain the dimer HO-GUm-SUC-TOB as a white solid. (228.7 mg, 96%) m / z(TOF-MS):calcd.1721.07, found 1723.10[M+H] + [Industrial Applicability]

[0296] According to the present invention, the by-production of phosphotriesters or phosphodiesters ("PO impurities") can be suppressed by the coexistence of a specific antioxidant in the stage of synthesizing a phosphite or phosphite diester, and the subsequent sulfurization reaction can be carried out more efficiently, making the present invention useful as a method for producing oligonucleotides having phosphorothioate sites.

[0297] This application is based on patent application No. 2019-065158 filed in Japan (filing date: March 28, 2019), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing an oligonucleotide having a phosphorothioate site, comprising the step of condensing a nucleoside, nucleotide or oligonucleotide (hereinafter sometimes referred to as "nucleic acid A") in which the 5'-position hydroxyl group is not protected and other groups may be protected with a protecting group used in nucleic acid synthesis or may be bound to a solid phase support, with a nucleoside, nucleotide or oligonucleotide (hereinafter sometimes referred to as "nucleic acid B") in which the 3'-position hydroxyl group or the 3'-position amino group has been modified by a method for forming a phosphite or phosphite diester and other groups may be protected with a protecting group used in nucleic acid synthesis, in the presence of an antioxidant to obtain a phosphite or phosphite diester (step (1)).

2. The method for producing an oligonucleotide having a phosphorothioate site according to claim 1, wherein the 3'-position hydroxyl group or the 3'-position amino group in the nucleic acid B is modified by a method selected from the group consisting of a phosphoramidite method, an H-phosphonate method, a dihalophosphine method and an oxazaphosphoridine method.

3. 3. A method for producing an oligonucleotide having a phosphorothioate site according to claim 1 or 2, further comprising a step (step (2)) of reacting the phosphite or phosphorous diester obtained in step (1) with a sulfurizing agent.

4. The method for producing an oligonucleotide having a phosphorothioate site according to any one of claims 1 to 3, wherein the antioxidant is selected from the group consisting of phosphorus-based antioxidants, sulfur-based antioxidants, and phosphorus-sulfur-based antioxidants.

5. The antioxidant, (1)P-(R) ３ (2)P-(OR) ３ (3)P(R) ２ (OR) (4)PR(OR) ２ (5)PH(O)(R) ２ (6)PH(O)(OR) ２ , and (7) PH(O)R(OR) (In the above formulas (1) to (7), R may be the same or different and independently represent an optionally substituted aryl group or a lower alkyl group, and two R may be bonded to each other to form a ring together with another adjacent atom; Here, the antioxidant may be a dimer in which the R moieties of the same or different antioxidants are bonded together. A phosphorus-based antioxidant selected from the group consisting of (8) R-S-R, and （９） 【Chemistry 1】 In the above formulas (8) and (9), R may be the same or different and each independently represents a lower alkyl group or an optionally substituted aryl group; Here, the antioxidant may be a dimer in which the R moieties of the same or different antioxidants are bonded together. A sulfur-based antioxidant selected from the group consisting of: (10)(OR) ２ P(S)-S-M-S-P(S)(OR) ２ (wherein R may be the same or different and independently represents a lower alkyl group or an optionally substituted aryl group, and M represents Zn, Ni or Cu; Here, the antioxidant may be a dimer in which the R moieties of the same or different antioxidants are bonded together. The method for producing an oligonucleotide having a phosphorothioate site according to any one of claims 1 to 3, wherein the phosphorus-sulfur antioxidant is selected from the group consisting of

6. The antioxidant, (1)P-(R) ３ (2)P-(OR) ３ (3)P(R) ２ (OR) (4) PR (OR) ２ , and (5) PH(O)R(OR) (In the above formulas (1) to (5), R may be the same or different and independently represent an optionally substituted aryl group or a lower alkyl group, and two R may be bonded to each other to form a ring together with another adjacent atom; Here, the antioxidant may be a dimer in which the R moieties of the same or different antioxidants are bonded together. A phosphorus-based antioxidant selected from the group consisting of （６） 【Chemistry 2】 In the above formula (6), R may be the same or different and each independently represents a lower alkyl group or an optionally substituted aryl group; Here, the antioxidant may be a dimer in which the R moieties of the same or different antioxidants are bonded together. The method for producing an oligonucleotide having a phosphorothioate site according to any one of claims 1 to 3, wherein the sulfur-based antioxidant is selected from the group consisting of

7. The method for producing an oligonucleotide having a phosphorothioate site according to any one of claims 1 to 3, wherein the antioxidant is selected from the group consisting of triphenylphosphine, methyldiphenylphosphine, triethyl phosphite, ethoxydiphenylphosphine, diethoxyphenylphosphine, 9,10-dihydro-9-oxa-10-phosphaphenanthrene 10-oxide (phosphorus-based antioxidants); and isobutylene sulfide (sulfur-based antioxidants).

8. The method for producing an oligonucleotide having a phosphorothioate site according to any one of claims 1 to 3, wherein the antioxidant is a phosphorus-based antioxidant.

9. The antioxidant is P-(R) ３ The method for producing the oligonucleotide having a phosphorothioate moiety according to any one of claims 1 to 3, wherein the phosphine is represented by the following formula:

10. The method for producing an oligonucleotide having a phosphorothioate site according to any one of claims 1 to 9, wherein nucleic acid A is a nucleoside, nucleotide or oligonucleotide in which the 5'-position hydroxyl group is not protected and other groups may be protected with a protecting group used in nucleic acid synthesis.

11. Nucleic acid A is a nucleoside, nucleotide or oligonucleotide in which the 5'-position hydroxyl group is not protected, and at least one group selected from the amino group and imino group of the nucleic acid base, the 2'-position hydroxyl group, the 3'-position hydroxyl group and the 3'-position amino group of the ribose residue, and the 3'-position hydroxyl group and the 3'-position amino group of the deoxyribose residue is protected with a protecting group that is not removable under acidic conditions but is removable under basic conditions (hereinafter, may be referred to as a "pseudo solid-phase protecting group"), and other groups may be further protected with a protecting group used in nucleic acid synthesis (hereinafter, may be referred to as "nucleic acid a"); or A nucleoside, nucleotide or oligonucleotide (hereinafter sometimes referred to as "nucleic acid α") in which the 5'-position hydroxyl group is not protected, one OH of the phosphate group at the 3'-terminus is replaced with -OLn1-OH (wherein Ln1 represents an organic group), the hydroxyl group of -OLn1-OH is protected with a protecting group that is not removable under acidic conditions but is removable under basic conditions, and other groups may be further protected with protecting groups used in nucleic acid synthesis, and nucleic acid B is a nucleoside, nucleotide or oligonucleotide in which the 3'-position hydroxyl group or the 3'-position amino group is in the form of a phosphite or or modified by a method for forming a phosphite diester, and the 5'-hydroxyl group is protected with a temporary protecting group removable under acidic conditions, and other groups may be further protected with a protecting group selected from a protecting group that is not removed under acidic conditions but is removable under basic conditions (hereinafter, may be referred to as a "pseudo solid-phase protecting group") and a protecting group used in nucleic acid synthesis (hereinafter, may be referred to as "nucleic acid b").

12. The method for producing an oligonucleotide having a phosphorothioate site according to any one of claims 1 to 11, wherein nucleic acid B is a nucleoside, nucleotide or oligonucleotide in which the 3'-position hydroxyl group has been modified by a method for forming a phosphite or a phosphorous diester and other groups may be protected with a protecting group used in nucleic acid synthesis.

13. The method for producing an oligonucleotide having a phosphorothioate site according to any one of claims 3 to 12, wherein the sulfurizing agent used in step (2) is selected from 5-[(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazole-3-thione (DDTT), 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent), 3H-1,2-benzodithiol-3-one, phenylacetyl disulfide (PADS), tetraethylthiuram disulfide (TETD), dipentamethylenethiuram tetrasulfide, 5-phenyl-3H-1,2,4-dithiazole-3-one (POS), 3-amino-1,2,4-dithiazole-5-thione (ADTT, xanthan hydride), N-[(2-cyanoethyl)thio]phthalimide and sulfur.

14. The method for producing an oligonucleotide having a phosphorothioate site according to any one of claims 1 to 13, further comprising a step of isolating an unprotected oligonucleotide having a phosphorothioate site after removing all protecting groups from the obtained oligonucleotide having a phosphorothioate site.

Images