Precipitation accelerator and precipitation method using the same

Abstract

The present invention provides a precipitation accelerator, which is an organic compound having one or more straight-chain aliphatic hydrocarbon groups with 10 or more carbon atoms, and the total number of carbon atoms of the aliphatic hydrocarbon groups is 20 or more. The precipitation accelerator of the present invention is used to precipitate an organic compound protected by an organic group having one or more aliphatic hydrocarbon groups with 10 or more carbon atoms from a solvent.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201680006676.9, application date January 21, 2016, and invention name “Precipitation accelerator and precipitation method using the same”. Technical Field

[0002] The present invention relates to a precipitation accelerator that can be used to precipitate an organic compound (eg, oligonucleotide, peptide, etc.) in a solution and a precipitation method using the same. Background Art

[0003] The synthetic method of oligonucleotide comprises phosphotriester method, H-phosphonate method, phosphoramidite method etc., and the solid phase synthesis (solid phase method) using phosphoramidite method is the most widely used method at present.In addition, as the synthetic method of peptide, solid phase synthesis is widely used.Solid phase method is advantageous with respect to speed aspect, because technology has been optimized and automation progress has been achieved.But the defective related thereto is: due to equipment limitation, scale amplification is limited, excessive reagent and starting material are used, and it is difficult to determine reaction progress state, analyze intermediate structure etc. in intermediate step.On the other hand, the synthetic method of oligonucleotide and peptide by liquid phase method has also been studied.But, due to complicated operation and low yield, it is difficult to synthesize long oligonucleotide and peptide quickly on a large scale.

[0004] In recent years, in an attempt to solve the respective defects of the solid phase method and the liquid phase method, in the liquid phase method, oligonucleotide synthesis methods using nucleosides protected by organic groups (pseudo solid phase protecting groups) having one or more long-chain aliphatic hydrocarbon groups (Patent Documents 1 and 2) and peptide synthesis methods using peptides having pseudo solid phase protecting groups (Patent Documents 3, 4 and 5) have been reported. In these synthesis methods, for example, the target product is recovered by adding a polar solvent to an oligonucleotide or peptide having a hydrophobic pseudo solid phase protecting group dissolved in a non-polar solvent to precipitate these compounds from a mixed solvent of a non-polar solvent and a polar solvent (solid-liquid separation).

[0005] [Literature List]

[0006] [Patent Document]

[0007] Patent Document 1: WO 2012 / 157723

[0008] Patent Document 2: WO 2014 / 189142

[0009] Patent Document 3: WO 2010 / 104169

[0010] Patent Document 4: WO 2010 / 113939

[0011] Patent Document 5: WO 2011 / 078295 Summary of the invention

[0012] [Problems to be solved by the invention]

[0013] In the liquid phase synthesis of organic compounds such as oligonucleotides, peptides, etc. using such pseudo-solid phase protecting groups, it is desired to more efficiently precipitate these compounds from the solvent to improve the recovery rate of the compounds.

[0014] The present invention has taken note of the above situation and aims to improve the recovery rate of an organic compound when the organic compound having a pseudo-solid-phase protecting group is precipitated from a solvent.

[0015] [Methods used to solve the problem]

[0016] The inventors of the present invention have conducted intensive research in an attempt to achieve the above object and have found that the precipitation of organic compounds having a hydrophobic group or a pseudo solid phase protecting group can be promoted and the recovery rate can be improved by using a specific precipitation promoter. The present invention based on these findings is as follows.

[0017] [1] A precipitation accelerator for precipitating an organic compound protected by an organic group from a solvent, the organic group having one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, wherein:

[0018] The precipitation accelerator is an organic compound having one or more straight-chain aliphatic hydrocarbon groups having 10 or more carbon atoms, wherein the total number of carbon atoms of the aliphatic hydrocarbon groups of the precipitation accelerator is 20 or more.

[0019] [2] The precipitation accelerator of [1] above, wherein the straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms in the precipitation accelerator is selected from the group consisting of straight-chain C 10-40 Alkyl and straight chain C 10-40 Alkenyl group.

[0020] [3] The precipitation accelerator of [1] or [2] above, wherein the aliphatic hydrocarbon group having 10 or more carbon atoms in the organic group is a straight chain type.

[0021] [4] The precipitation accelerator of [1] or [2] above, wherein the aliphatic hydrocarbon group having 10 or more carbon atoms in the organic group is selected from a linear C 10-40 Alkyl and straight chain C 10-40 Alkenyl group.

[0022] [5] The precipitation accelerator of any one of [1] to [4] above, wherein the precipitation accelerator is

[0023] (1) an organic compound having one or more structures represented by formula (G); or

[0024] (2) having one or more linear C 10-40 Alkyl, and further optionally substituted C 1-10 Alkanes,

[0025] Wherein, the formula (G) is:

[0026]

[0027] In the formula,

[0028] R 1 Each independently represents a straight chain C 10-40 alkyl;

[0029] X 1 each independently represents a single bond, -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0030] Ring A 1 represents an optionally substituted C 3-14 hydrocarbon ring;

[0031] n represents an integer of 1 to 4.

[0032] [6] The precipitation accelerator of any one of [1] to [4] above, wherein the precipitation accelerator is

[0033] (1) a compound represented by formula (I); or

[0034] (2) having one or more linear C 10-40 Alkyl, and further optionally substituted C 1-10 Alkanes,

[0035] Wherein, the formula (I) is:

[0036]

[0037] In the formula,

[0038] R 1 Each independently represents a straight chain C 10-40 alkyl;

[0039] X 1each independently represents a single bond, -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0040] Ring A 1 and Ring B 1 Each independently represents an optionally substituted C 3-14 hydrocarbon ring;

[0041] X 2 Each independently represents -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -、-(CH2) p -OC(=O)-(CH2) q -、-(CH2) p -C(=O)NH-(CH2) q -or-(CH2) p -NHC(=O)-(CH2) q -, wherein p and q each independently represent an integer from 0 to 3;

[0042] X 3 Represents a single bond, -(CH2) r -O-, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O-, -(CH2) r -OC(=O)-, -(CH2) r -C(=O)NH- or -(CH2) r -NHC(=O)-, wherein r represents an integer from 0 to 3;

[0043] R 2 represents a hydrogen atom, an optionally substituted C 3-8 Cycloalkyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 6-14 aryl, optionally substituted monocyclic heterocyclic or tri(C 1-6 Alkyl)silyl;

[0044] n and m each independently represent an integer from 1 to 4;

[0045] When m is 1, m' represents 0 or 1; when m is 2, 3 or 4, m' represents 1.

[0046] [7] The precipitation accelerator of [6] above, wherein R 2 is a hydrogen atom, an optionally substituted C 3-8 Cycloalkyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 6-14 aryl or an optionally substituted monocyclic heterocyclic group.

[0047] [8] The precipitation accelerator according to any one of [1] to [4] above, wherein the precipitation accelerator is a compound represented by formula (II),

[0048]

[0049] In the formula,

[0050] R 1 Each independently represents a straight chain C 10-40 alkyl;

[0051] X 1 each independently represents a single bond, -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0052] Ring A 2 and Ring B 1 Each independently represents an optionally substituted C 3-14 hydrocarbon ring;

[0053] X 2 Each independently represents -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -、-(CH2) p -OC(=O)-(CH2) q -、-(CH2) p -C(=O)NH-(CH2) q -or-(CH2) p -NHC(=O)-(CH2) q -, wherein p and q each independently represent an integer from 0 to 3;

[0054] X3 Represents a single bond, -(CH2) r -O-, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O-, -(CH2) r -OC(=O)-, -(CH2) r -C(=O)NH- or -(CH2) r -NHC(=O)-, wherein r represents an integer from 0 to 3;

[0055] R 2 represents a hydrogen atom, an optionally substituted C 3-8 Cycloalkyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 6-14 aryl, optionally substituted monocyclic heterocyclic or tri(C 1-6 Alkyl)silyl;

[0056] m represents an integer from 1 to 4;

[0057] When m is 1, m' represents 0 or 1; when m is 2, 3 or 4, m' represents 1.

[0058] [9] The precipitation accelerator of [8] above, wherein R 2 is a hydrogen atom, an optionally substituted C 3-8 Cycloalkyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 6-14 aryl or an optionally substituted monocyclic heterocyclic group.

[0059]

[10] The precipitation accelerator according to any one of [5] to [9] above, wherein C 3-14 The hydrocarbon ring is selected from a benzene ring and a cyclohexane ring.

[0060]

[11] The precipitation accelerator according to any one of [1] to

[10] above, wherein the organic group is a group represented by formula (III),

[0061] **LYZ (III)

[0062] In the formula,

[0063] ** Indicates the bonding position to the protected group;

[0064] L represents a single bond, or a group represented by formula (a1) or (a1'):

[0065]

[0066] In the formula,

[0067] * represents the bonding position with Y;

[0068] ** Same as the above definition;

[0069] R 8 and R 9 Each independently represents C 1-22 Hydrocarbon;

[0070] L1 indicates divalent C 1-22 a hydrocarbon group; and

[0071] L2 represents a single bond, or ** C(=O)N(R 2’ )-R 1’ -N(R 3 ) *** The group shown in the formula ** represents the bonding position with L1, *** Indicates the bonding position with C=O, R 1’ Represents C 1-22 Alkylene, R 2’ and R 3 Each independently represents a hydrogen atom or a C 1-22 Alkyl, or R 2’ and R 3 optionally forming a ring together;

[0072] Y represents a single bond, an oxygen atom or NR, wherein R represents a hydrogen atom, an alkyl group or an aralkyl group; and

[0073] Z represents a group represented by formula (a2), formula (a2') or formula (a2"):

[0074]

[0075] In the formula,

[0076] * Indicates the bonding position;

[0077] R 4 is a hydrogen atom, or when R b When it is a group represented by the following formula (a3), R 4 Optionally with R of ring C 6 Together they represent a single bond or -O-, and together with ring A or ring B and ring C form a condensed ring;

[0078] k Qs each independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0079] k R 5 Each independently represents a hydrocarbon group to which an aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group;

[0080] k represents an integer from 1 to 4;

[0081] Ring A and Ring B each independently have k QR 5 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C optionally substituted by halogen atoms 1-6 Alkoxy;

[0082] R a represents a hydrogen atom, or a phenyl group which is optionally substituted by a halogen atom; and

[0083] R b represents a hydrogen atom, or a group represented by formula (a3):

[0084]

[0085] In the formula, * Indicates the bonding position;

[0086] j represents an integer from 0 to 4;

[0087] j Qs each independently represent the same meaning as above;

[0088] j R 7 Each independently represents a hydrocarbon group to which an aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group;

[0089] R 6 represents a hydrogen atom, or optionally connected to R of ring A or ring B 4 together represent a single bond or -O-, and together with ring A or ring B and ring C form a condensed ring; and

[0090] In addition to having j QR 7 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C optionally substituted by halogen atoms 1-6 Alkoxy, or

[0091] R a and R b Together they form an oxo group.

[0092]

[12] The precipitation accelerator of

[11] above, wherein Z is a group represented by formula (a2) or formula (a2′), the condensed ring is a fluorene ring or a xanthene ring, and k R 5 Each of the j R 7 are each independently a hydrocarbon group to which a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group, and R a is a hydrogen atom, or R a and R b together to form a carbonyl group.

[0093]

[13] The precipitation accelerator of

[11] or

[12] above, wherein L is a group represented by formula (a1), L1 represents a divalent C 1-22 A hydrocarbon group, and L2 represents a single bond.

[0094]

[14] The precipitation accelerator of the above-mentioned

[11] or

[12] , wherein L is a group represented by formula (a1′), L1 represents a phenylene group, and L2 represents a single bond.

[0095]

[15] The precipitation accelerator of any one of

[11] to

[14] above, wherein Y is an oxygen atom.

[0096]

[16] The precipitation accelerator according to any one of

[11] to

[15] above, wherein Z is a group represented by formula (a2), R 4 A hydrogen atom.

[0097]

[17] The precipitation accelerator according to any one of

[11] to

[16] above, wherein Z is a group represented by formula (a2), R a and R b A hydrogen atom.

[0098]

[18] The precipitation accelerator of

[11] or

[12] above, wherein L and Y are single bonds, Z is a group represented by formula (a2), and R 4 is a hydrogen atom, and R a and R b together to form a carbonyl group.

[0099]

[19] The precipitation promoter of any one of [1] to

[18] above, wherein the organic compound protected by an organic group is a nucleoside, nucleotide or oligonucleotide optionally further protected by a protecting group used in nucleic acid synthesis, or an amino acid or peptide optionally further protected by a protecting group used in peptide synthesis.

[0100]

[20] The precipitation accelerator of any one of [1] to

[18] above, wherein the organic compound protected by an organic group is a nucleoside, nucleotide or oligonucleotide optionally further protected by a protecting group used in nucleic acid synthesis.

[0101]

[21] The precipitation accelerator of any one of [1] to

[18] above, wherein the organic compound protected by an organic group is a nucleoside or an oligonucleotide,

[0102] In the nucleoside or oligonucleotide, at least one group selected from the amino and imino groups of the nucleic acid base, the 2' and 3' hydroxyl groups of the ribose residue, and the 3' hydroxyl group of the deoxyribose residue is protected by an organic group, and the other groups are optionally further protected by a protecting group used in nucleic acid synthesis.

[0103]

[22] The precipitation accelerator of any one of [1] to

[21] above, wherein the solvent is a solvent containing a polar solvent.

[0104]

[23] The precipitation accelerator of the above-mentioned

[22] , wherein the solvent containing the polar solvent is a mixed solvent of a polar solvent and a non-polar solvent.

[0105]

[24] The precipitation accelerator of

[22] or

[23] above, wherein the polar solvent is acetonitrile.

[0106]

[25] The precipitation accelerator of any one of [1] to

[24] above, wherein the organic compound protected by an organic group is precipitated by using the precipitation accelerator in an amount of 0.1 molar equivalent or more relative to the organic compound protected by an organic group.

[0107]

[26] A precipitation mixture comprising the precipitation accelerator of any one of [1] to

[25] above, and an organic compound protected by an organic group having one or more aliphatic hydrocarbon groups having 10 or more carbon atoms.

[0108]

[27] The precipitation mixture of

[26] above, wherein the aliphatic hydrocarbon group having 10 or more carbon atoms in the organic group is a straight chain type.

[0109]

[28] A method for precipitating an organic compound protected by an organic group having one or more aliphatic hydrocarbon groups having 10 or more carbon atoms from a solvent, using the precipitation accelerator of any one of [1] to

[25] above.

[0110]

[29] The method of

[28] above, wherein the solvent is a solvent containing a polar solvent.

[0111]

[30] The method of

[29] above, wherein the polar solvent is acetonitrile.

[0112]

[31] The method according to any one of

[28] to

[30] above, wherein the aliphatic hydrocarbon group having 10 or more carbon atoms in the organic group is a straight chain type.

[0113]

[32] A method for preparing an oligonucleotide, the method comprising: adding a polar solvent to a reaction solution containing an oligonucleotide and the precipitation accelerator of any one of [1] to

[25] in a non-polar solvent, and separating a precipitation mixture containing the oligonucleotide and the precipitation accelerator from the reaction solution;

[0114] In the oligonucleotide, at least one group is protected by an organic group having one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, and other groups are optionally further protected by a protecting group used in nucleic acid synthesis.

[0115]

[33] The preparation method of the above-mentioned

[32] , wherein the polar solvent is acetonitrile.

[0116]

[34] The production method of

[32] or

[33] above, wherein the aliphatic hydrocarbon group having 10 or more carbon atoms in the organic group is a straight chain type.

[0117]

[35] The method for preparing the compound according to any one of

[32] to

[34] above, wherein the method is carried out by a phosphoramidite method.

[0118]

[36] A method for preparing an oligonucleotide, which is a method for preparing an oligonucleotide using a phosphoramidite method, wherein the phosphoramidite method comprises performing a preparation cycle comprising the following steps (1) to (3) once or repeatedly for a plurality of times,

[0119] wherein the first cycle includes the following step (4), each cycle includes the following step (5), and each cycle except the final cycle includes the following step (6),

[0120] (1) adding an acid to a reaction solution containing a nucleoside or an oligonucleotide in a non-polar solvent to deprotect the temporary protecting group of the 5'-hydroxyl group, and neutralizing the solution with a base to obtain a reaction solution containing a free form of the 5'-hydroxyl group,

[0121] In the nucleoside or oligonucleotide, at least one group selected from the amino and imino groups of the nucleic acid bases, the 2' and 3' hydroxyl groups of the ribose residue, and the 3' hydroxyl group of the deoxyribose residue is protected by an organic group having one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, the 5' hydroxyl group is protected by a temporary protecting group removable under acidic conditions, and the other groups are optionally further protected by protecting groups used in nucleic acid synthesis;

[0122] (2) a step of adding a nucleoside or an oligonucleotide to a reaction solution containing a free 5'-hydroxyl group in a non-polar solvent to obtain a reaction solution containing a triester phosphite compound,

[0123] In the nucleoside or oligonucleotide, the 3'-hydroxyl group is phosphoramidated, and the 5'-hydroxyl group is protected by a temporary protecting group that can be removed under acidic conditions, and other groups are optionally further protected by protecting groups used in nucleic acid synthesis;

[0124] (3) adding an oxidizing agent or a sulfurizing agent to a reaction solution containing a triester phosphite compound in a non-polar solvent to obtain a reaction solution containing an oligonucleotide,

[0125] In the oligonucleotide, at least one group selected from the group consisting of the amino and imino groups of the nucleic acid bases, the 2' and 3' hydroxyl groups of the ribose residue, and the 3' hydroxyl group of the deoxyribose residue is protected by an organic group having one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, and the 5' hydroxyl group is protected by a temporary protecting group removable under acidic conditions, and the other groups are optionally further protected by protecting groups used in nucleic acid synthesis;

[0126] (4) a step of adding the precipitation accelerator of any one of [1] to

[25] to the reaction solution at any time before step (1), between steps (1) and (2), between steps (2) and (3), and after step (3);

[0127] (5) after step (4) and at any time between steps (1) and (2), between steps (2) and (3), and after step (3), a step of adding a polar solvent to the reaction solution containing the precipitation accelerator to separate a precipitation mixture containing the 5'-hydroxyl free body, the triester phosphite compound or the oligonucleotide, and the precipitation accelerator from the reaction solution;

[0128] (6) A step of adding a non-polar solvent to the precipitate mixture obtained in step (5) to prepare a reaction solution.

[0129]

[37] The preparation method of the above-mentioned

[36] , wherein the polar solvent is acetonitrile.

[0130]

[38] The production method of

[36] or

[37] above, wherein the aliphatic hydrocarbon group having 10 or more carbon atoms in the organic group is a straight chain type.

[0131]

[39] The method of any one of

[36] -

[38] above, wherein steps (5) and (6) are performed after step (3).

[0132]

[40] The method of any one of

[36] to

[39] above, further comprising the following step (7):

[0133] (7) A step of removing all protecting groups of the oligonucleotide and isolating the oligonucleotide.

[0134]

[41] The method of any one of

[36] to

[40] above, wherein the non-polar solvent is a solvent selected from the group consisting of halogen solvents, aromatic solvents, ester solvents, aliphatic solvents, and combinations thereof.

[0135] [Effects of the Invention]

[0136] The use of the precipitation accelerator of the present invention can promote the precipitation of an organic compound having a pseudo solid phase protecting group (which is a hydrophobic group) from a solvent and improve its recovery rate. DETAILED DESCRIPTION

[0137] [the term]

[0138] Unless otherwise specified in the text, any technical terms and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. Any methods and materials similar or equivalent to those described in this specification can be used in the implementation or test of the present invention, and preferred methods and materials are recorded hereinafter. All publications and patents cited in this specification are incorporated herein by reference to describe and disclose the construction products and methodology described in, for example, publications useful for the invention.

[0139] In the present specification, "nucleoside" which is a constituent unit of oligonucleotide refers to a compound in which a nucleic acid base is bonded to the 1' position of a sugar (e.g., ribose or deoxyribose in which the carbon atom at the 2nd position and the carbon atom at the 4th position of 2-deoxyribose, ribose, a ribose ring or a deoxyribose ring are bonded via a divalent organic group, etc.) by N-glycosylation. Examples of ribose or deoxyribose in which the carbon atom at the 2nd position and the carbon atom at the 4th position of a ribose ring or a deoxyribose ring are bonded via a divalent organic group include the following compounds.

[0140]

[0141] In the present specification, "nucleotide" refers to a compound in which a phosphate group is bonded to a nucleoside.

[0142] In the present specification, "oligonucleotide" refers to a compound in which one or more nucleotides are bonded to nucleosides. Although the number of nucleosides of the oligonucleotide in the present invention is not particularly limited, it is preferably 3-50, more preferably 5-30.

[0143] In the present specification, "nucleoside" and "nucleotide" include morpholino nucleosides and morpholino nucleotides having a morpholine residue instead of a ribose residue or a deoxyribose residue.

[0144] In the present specification, "morpholino nucleoside" is a compound represented by the following formula (1). In the present specification, the morpholino nucleoside represented by formula (1) is referred to as morpholino nucleoside (1). Compounds represented by other formulae can also be referred to in the same manner.

[0145]

[0146] wherein base represents an optionally protected nucleic acid base.

[0147] Morpholino nucleoside (1) can be prepared by a method known per se (e.g., the method described in WO91 / 09033A1) or a method analogous thereto. Specifically, as shown in the following scheme, the corresponding ribonucleoside (2) is oxidatively opened using sodium periodate or the like to form the corresponding 2',3'-dialdehyde (3), the dialdehyde (3) is ring-closed using ammonia to form a 2',3'-dihydroxymorpholino nucleoside (4), and the dihydroxymorpholino nucleoside (4) is reduced using a reducing agent (e.g., sodium cyanoborohydride, sodium triacetoxyborohydride, etc.), thereby obtaining morpholino nucleoside (1).

[0148]

[0149] In this specification, the position numbers (1', 2', etc.) of the morpholino nucleoside correspond to the position numbers of the carbon atoms of the ribose of the raw material ribonucleoside (2). In this specification, according to the convention of nucleic acid chemistry, the morpholino nucleoside at the end of the morpholino oligonucleotide having the free 5' hydroxyl group is referred to as the "5'-end", and the morpholino nucleoside at the opposite end is referred to as the "3'-end".

[0150] In this specification, "nucleic acid base" is not particularly limited, as long as it can be used for synthetic nucleic acid, and includes, for example, pyrimidine bases such as cytosine base, uracil base, thymine base, etc., and purine bases such as adenine base, guanine base, etc. "Optionally protected nucleic acid base" refers to, for example, in adenine base, guanine base or cytosine base (which is a nucleic acid base with an amino group), the amino group can be protected, preferably wherein the amino group of the nucleic acid base is protected by a protecting group that can tolerate the deprotection conditions of the 5' position of the nucleotide or the deprotection conditions of the morpholine ring nitrogen atom of the morpholino nucleotide. The "protective group of amino group" is not particularly limited, and examples thereof include the protecting groups described in Greene's Protective Groups in Organic Synthesis, 4th edition, Wiley-Interscience, 2006, etc. Specific examples of the "amino protecting group" include pivaloyl, pivaloyloxymethyl, trifluoroacetyl, phenoxyacetyl, 4-isopropylphenoxyacetyl, 4-tert-butylphenoxyacetyl, acetyl, benzoyl, isobutyryl, dimethylformamidinyl, 9-fluorenylmethoxycarbonyl, etc. Among them, phenoxyacetyl, 4-isopropylphenoxyacetyl, acetyl, benzoyl, isobutyryl and dimethylformamidinyl are preferred. In addition, the carbonyl group of the nucleic acid base is optionally protected, for example, by reacting 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-nitrophenylethanol, 2-(methylsulfonyl)ethanol, 2-(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol, dimethylcarbamoyl chloride, diethylcarbamoyl chloride, ethylphenylcarbamoyl chloride, 1-pyrrolidinecarbonyl chloride, 4-morpholinecarbonyl chloride, diphenylcarbamoyl chloride, etc. to protect the carbonyl group. In some cases, there is no need to specifically introduce a protecting group for the carbonyl group. In addition, in addition to the above-mentioned groups, the "nucleic acid base" also includes modified nucleic acid bases (for example, 8-bromoadenine, 8-bromoguanine, 5-bromocytosine, 5-iodocytosine, 5-bromouracil, 5-iodouracil, 5-fluorouracil, 5-methylcytosine, 8-oxoguanine, hypoxanthinyl, etc.), which are nucleic acid bases substituted by any 1 to 3 substituents (for example, halogen atoms, alkyl groups, aralkyl groups, alkoxy groups, acyl groups, alkoxyalkyl groups, hydroxyl groups, amino groups, monoalkylamino groups, dialkylamino groups, carboxyl groups, cyano groups, nitro groups, etc.) at any position.

[0151] In the present specification, the "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

[0152] In the present specification, examples of "alkyl (group)" include straight or branched alkyl groups having one or more carbon atoms. When the carbon number is not particularly limited, it is preferably C 1-10 Alkyl, more preferably C 1-6 When the number of carbon atoms is not particularly limited, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like are preferred, and methyl and ethyl are particularly preferred.

[0153] In this manual, “C a-b " means that the number of carbon atoms is greater than or equal to a and less than or equal to b (a and b each represent an integer).

[0154] In the present specification, examples of the "aralkyl (group)" include C 7-20 Arylalkyl, preferably C 7-16 Arylalkyl (C 6-10 Aryl-C 1-6 Specific preferred examples include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-naphthylethyl, 1-naphthylpropyl, etc., and benzyl is particularly preferred.

[0155] In the present specification, examples of the "alkoxy (group)" include alkoxy groups having one or more carbon atoms. When the carbon number is not particularly limited, it is preferably C 1-10 Alkoxy, more preferably C 1-6 Alkoxy. When the number of carbon atoms is not particularly limited, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy and the like are preferred, and methoxy and ethoxy are particularly preferred.

[0156] In the present specification, examples of "acyl (group)" include linear or branched C 1-6 Alkanoyl, C 7-13 Aroyl, etc. Specific examples thereof include formyl, acetyl, n-propionyl, isopropionyl, n-butyryl, isobutyryl, pivaloyl, valeryl, hexanoyl, benzoyl, naphthoyl, levulinyl, etc., each of which is optionally substituted.

[0157] In the present specification, the "alkenyl (group)" is preferably a straight chain or branched C 2-6 Examples thereof include vinyl, 1-propenyl, allyl, isopropenyl, butenyl, isobutenyl, etc. Among them, C 2-4 Alkenyl.

[0158] In the present specification, the "alkynyl (group)" preferably includes C 2-6Preferred examples thereof include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, etc. Among them, C 2-4 Alkynyl.

[0159] In the present specification, "cycloalkyl (group)" refers to a cyclic alkyl group, examples of which include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. Among them, C 3-6 Examples of the cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, with cyclohexyl being particularly preferred.

[0160] In the present specification, "aryl (group)" refers to an aromatic monocyclic hydrocarbon group or a polycyclic (condensed) hydrocarbon group. Specific examples thereof include C 6-14 Aryl groups include phenyl, 1-naphthyl, 2-naphthyl, biphenyl, 2-anthryl, etc. Among them, C 6-10 The aryl group is particularly preferably a phenyl group.

[0161] In the present specification, examples of the "hydrocarbon group" include aliphatic hydrocarbon groups, aromatic aliphatic hydrocarbon groups, monocyclic saturated hydrocarbon groups, aromatic hydrocarbon groups, etc. Specific examples thereof include monovalent groups such as alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, aryl groups, aralkyl groups, etc., and divalent groups derived therefrom.

[0162] In the present specification, the three alkyl groups in the "trialkylsilyl (group)" may be the same or different. The alkyl groups may be independently straight chain or branched. As the "trialkylsilyl (group)", preferably tri(C 1-6 As the silyl group (alkyl), for example, there can be mentioned trimethylsilyl, triethylsilyl, triisopropylsilyl and tert-butyldimethylsilyl.

[0163] In the present specification, the "alkylene (group)" may be any of a straight chain and a branched chain. As the "alkylene (group)", an alkylene group having one or more carbon atoms may be mentioned. When the carbon number is not particularly limited, it is preferably C 1-10 Alkylene, more preferably C 1-6 Specific preferred examples include methylene, ethylene, propylene, butylene, pentylene and hexylene, with methylene and ethylene being particularly preferred.

[0164] In the present specification, examples of the “linking group” include —O—, —C(═O)—, —C(═O)O—, —OC(═O)—, —C(═O)NH—, —NHC(═O)—, —S—, —SO—, —SO2—, —Si(R)(R′)O—, —Si(R)(R′)— (R and R′ are each independently a hydrogen atom or a C1-22 Hydrocarbon) etc.

[0165] In the present specification, the “substituent” in “optionally substituted” includes the above-mentioned halogen atoms, alkyl groups, aralkyl groups, alkoxy groups, acyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, aryl groups, as well as hydroxyl groups, nitro groups, cyano groups, guanidino groups, carboxyl groups, alkoxycarbonyl groups (the alkoxy moiety is the same as the above-mentioned alkoxy group), sulfo groups (sulfo group), phosphoryl groups (phospho group), alkylthio groups (the alkyl moiety is the same as the above-mentioned alkyl group), alkylsulfinyl groups (the alkyl moiety is the same as the above-mentioned alkyl group), alkylsulfonyl groups (the alkyl moiety is the same as the above-mentioned alkyl group), amino groups, monoalkylamino groups (the alkyl moiety is the same as the above-mentioned alkyl group), dialkylamino groups (the alkyl moiety is the same as the above-mentioned alkyl group), carbonyl groups (oxo group), acylamino groups (the acyl moiety is the same as the above-mentioned acyl group), and the like.

[0166] In this manual, “C 3-14 Hydrocarbon ring (including "optionally substituted C 3-14 The "C" in the hydrocarbon ring 3-14 Examples of "hydrocarbon ring" include C 3-10 Cycloalkanes, C 3-10 Cycloolefins, C 6-14 Aromatic hydrocarbon ring.

[0167] The "C 3-10 Examples of "cycloalkanes" include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane.

[0168] The "C 3-10 Examples of "cycloolefins" include cyclopropene, cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclooctene.

[0169] The "C 6-14 Examples of the "aromatic hydrocarbon ring" include benzene, naphthalene.

[0170] In the present specification, examples of the “monocyclic heterocyclic group” (including the “monocyclic heterocyclic group” in the “optionally substituted monocyclic heterocyclic group”) include a monocyclic aromatic heterocyclic group and a monocyclic non-aromatic heterocyclic group, each containing 1 to 4 hetero atoms selected from a nitrogen atom, a sulfur atom and an oxygen atom as atoms constituting the ring other than carbon atoms.

[0171] Examples of the “monocyclic aromatic heterocyclic group” include a 5- or 6-membered monocyclic aromatic heterocyclic group such as thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, triazinyl and the like.

[0172] Examples of the “monocyclic non-aromatic heterocyclic group” include a 3- to 8-membered monocyclic non-aromatic heterocyclic group such as aziridinyl, oxiranyl, thiirane, azetidinyl, oxetanyl, thietanyl, tetrahydrothiophenyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, oxazolinyl, oxazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl, thiazolidinyl, tetrahydroisothiazolyl, tetrahydrooxazolyl, tetrahydroisoxazolyl, piperidinyl, piperazinyl, tetrahydropyridinyl, dihydropyridinyl, dihydrothiopyranyl, tetrahydropyrimidinyl, tetrahydropyridazinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl (e.g., morpholin-4-yl), thiomorpholinyl, azepanyl, diazepanyl, azepanyl, azepinyl, oxepanyl, azocanyl, diazocanyl, and the like.

[0173] [Precipitation accelerator]

[0174] The precipitation accelerator of the present invention is an organic compound having one or more straight-chain aliphatic hydrocarbon groups having 10 or more carbon atoms, and the total number of carbon atoms of the aliphatic hydrocarbon groups is 20 or more, and the precipitation accelerator is used to precipitate an organic compound protected by an organic group having one or more aliphatic hydrocarbon groups having 10 or more carbon atoms (hereinafter sometimes referred to as "pseudo solid phase protecting group of the present invention") from a solvent. The precipitation accelerator of the present invention may be used alone, or two or more thereof may be used in combination.

[0175] The above-mentioned solvent is preferably a solvent containing a polar solvent, and more preferably a mixed solvent of a polar solvent and a non-polar solvent. The above-mentioned polar solvent is preferably acetonitrile.

[0176] The total number of carbon atoms of the straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms of the precipitation accelerator of the present invention needs to be 20 or more, preferably 22 or more. When the total number of carbon atoms is less than 20, the organic compound protected by the pseudo-solid phase protecting group in the liquid phase cannot be sufficiently precipitated, and the recovery rate of the organic compound is reduced. Although the upper limit is not particularly limited, the total number of carbon atoms is preferably not more than 220, more preferably not more than 100, and further preferably not more than 70.

[0177] The straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms in the precipitation accelerator of the present invention is preferably selected from the group consisting of straight-chain C 10-40 Alkyl and straight chain C 10-40 Alkenyl groups, more preferably straight chain C 10-40 Alkyl, more preferably a straight chain C 10-30 Alkyl, particularly preferably straight chain C 12-28 Alkyl, most preferably straight chain C 14-26Alkyl group. The number of the linear aliphatic hydrocarbon groups having 10 or more carbon atoms in the precipitation accelerator of the present invention is preferably 1-12, more preferably 1-9.

[0178] One embodiment of the precipitation accelerator of the present invention is an organic compound having one or more structures represented by the following formula (G):

[0179]

[0180] In the formula,

[0181] R 1 Each independently represents a straight chain C 10-40 alkyl;

[0182] X 1 each independently represents a single bond, -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0183] Ring A 1 represents an optionally substituted C 3-14 hydrocarbon ring;

[0184] n represents an integer of 1 to 4.

[0185] In formula (G), X 1 The bonding direction of X corresponds to the direction of the above chemical formula. 1 When it is -C(=O)O-, it is on the left side of -C(=O)O- and R 1 Bonded to the right side of -C(=O)O- and ring A 1 Bonding.

[0186] When there are multiple R 1 When , they may be the same or different. The same applies to X 1 .

[0187] A preferred embodiment of the precipitation accelerator of the present invention is a compound represented by the following formula (I). "Compound represented by formula (I)" is sometimes abbreviated as "Compound (I)". Compounds represented by other formulae may also be abbreviated similarly.

[0188]

[0189] In the formula,

[0190] R 1 Each independently represents a straight chain C 10-40 alkyl;

[0191] X 1each independently represents a single bond, -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0192] Ring A 1 and Ring B 1 Each independently represents an optionally substituted C 3-14 hydrocarbon ring;

[0193] X 2 Each independently represents -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -、-(CH2) p -OC(=O)-(CH2) q -、-(CH2) p -C(=O)NH-(CH2) q -or-(CH2) p -NHC(=O)-(CH2) q -, wherein p and q each independently represent an integer from 0 to 3;

[0194] X 3 Represents a single bond, -(CH2) r -O-, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O-, -(CH2) r -OC(=O)-, -(CH2) r -C(=O)NH- or -(CH2) r -NHC(=O)-, wherein r represents an integer from 0 to 3;

[0195] R 2 represents a hydrogen atom, an optionally substituted C 3-8 Cycloalkyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 6-14 aryl, optionally substituted monocyclic heterocyclic or tri(C 1-6 Alkyl) silyl (preferably a hydrogen atom, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 6-14 aryl or optionally substituted monocyclic heterocyclyl);

[0196] n and m each independently represent an integer from 1 to 4;

[0197] When m is 1, m' represents 0 or 1; when m is 2, 3 or 4, m' represents 1.

[0198] In formula (I), X 1 , X 2 and X 3 The bonding directions of X correspond to the directions of the above chemical formula. 3 -(CH2) r -O-, it is in -(CH2) r -O- Left side with ring B 1 Bonded and in -(CH2) r -O- on the right side with R 2 Bonding.

[0199] When there are multiple R 1 When , they may be the same or different. The same applies to X 1 , Ring A 1 and X 2 .

[0200] A more preferred embodiment of the precipitation accelerator of the present invention is a compound represented by the following formula (II).

[0201]

[0202] In the formula,

[0203] R 1 Each independently represents a straight chain C 10-40 alkyl;

[0204] X 1 each independently represents a single bond, -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0205] Ring A 2 and Ring B 1 Each independently represents an optionally substituted C 3-14 hydrocarbon ring;

[0206] X 2 Each independently represents -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2)p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -、-(CH2) p -OC(=O)-(CH2) q -、-(CH2) p -C(=O)NH-(CH2) q -or-(CH2) p -NHC(=O)-(CH2) q -, wherein p and q each independently represent an integer from 0 to 3;

[0207] X 3 Represents a single bond, -(CH2) r -O-, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O-, -(CH2) r -OC(=O)-, -(CH2) r -C(=O)NH- or -(CH2) r -NHC(=O)-, wherein r represents an integer from 0 to 3;

[0208] R 2 represents a hydrogen atom, an optionally substituted C 3-8 Cycloalkyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 6-14 aryl, optionally substituted monocyclic heterocyclic or tri(C 1-6 Alkyl) silyl (preferably a hydrogen atom, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 6-14 aryl or optionally substituted monocyclic heterocyclyl);

[0209] m represents an integer from 1 to 4;

[0210] When m is 1, m' represents 0 or 1; when m is 2, 3 or 4, m' represents 1.

[0211] In formula (II), X 1 , X 2 and X 3 The bonding directions of X correspond to the directions of the above chemical formula. 3-(CH2) r -O-, it is in -(CH2) r -O- Left side with ring B 1 Bonded and in -(CH2) r -O- on the right side with R 2 Bonding.

[0212] When there are multiple R 1 When , they may be the same or different. The same applies to X 1 When there are multiple rings A in formula (II) 2 When , they may be the same or different. The same applies to X 2 .

[0213] When there are multiple R 1 When , they may be the same or different. 1 Preferably, each of the following is a straight chain C 10-30 Alkyl, more preferably straight chain C 12-28 Alkyl, more preferably a straight chain C 14-26 alkyl.

[0214] In one embodiment of the present invention, R 2 Preferably a hydrogen atom, an optionally substituted C 3-8 Cycloalkyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 6-14 aryl or an optionally substituted monocyclic heterocyclic group, more preferably a hydrogen atom, an optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl or optionally substituted C 6-14 Aryl, more preferably a hydrogen atom or an optionally substituted C 1-6 Alkyl, particularly preferably a hydrogen atom or a C 1-6 alkyl.

[0215] In another embodiment of the present invention, R 2 Preferably a hydrogen atom, an optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl or optionally substituted C 6-14 aryl, optionally substituted 3-8 membered monocyclic non-aromatic heterocyclic group or tri(C 1-6 alkyl) silyl group, more preferably a hydrogen atom or an optionally substituted C 1-6 Alkyl or 3-8 membered monocyclic non-aromatic heterocyclic group or tri(C 1-6Alkyl) silyl group, more preferably a hydrogen atom, optionally replaced by C 1-6 Alkanoylamino (e.g., acetylamino) substituted C 1-6 alkyl or a 3-8 membered monocyclic non-aromatic heterocyclic group (e.g., morpholin-4-yl) or a tri(C 1-6 alkyl)silyl (e.g., triisopropylsilyl).

[0216] X 1 Each independently is preferably a single bond, -O-, -C(=O)-, -C(=O)O- or -OC(=O)-, more preferably -O-, -C(=O)O- or -OC(=O)-, further preferably -O-.

[0217] X 2 Each is independently preferably -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)-(CH2) q -(p and q are each independently an integer of 0-3), more preferably -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)-(CH2) q -(p and q are each independently an integer of 0-3), more preferably a single bond, -(CH2) p -O-(CH2) q -, -C(=O)-, -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)- (p and q are each independently an integer of 0 or 1).

[0218] X 3 Preferably, it is a single bond, -(CH2) r -O-, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O- or -(CH2)r -OC(=O)- (r is an integer from 0 to 3), more preferably a single bond, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O- or -(CH2) r -OC(=O)- (r is an integer from 0 to 3), more preferably a single bond, -C(=O)-, -C(=O)O- or -OC(=O)-.

[0219] n is preferably an integer of 1-3.

[0220] m is preferably an integer of 1-3, more preferably 1 or 3.

[0221] C 3-14 The hydrocarbon ring is preferably selected from a benzene ring and a cyclohexane ring, and is more preferably a benzene ring.

[0222] Preferred compounds (I) are compounds wherein:

[0223] R 1 Each independently is a straight chain C 10-30 alkyl;

[0224] X 1 are each independently a single bond, -O-, -C(=O)-, -C(=O)O- or -OC(=O)-;

[0225] Ring A 2 and Ring B 1 Each is independently a benzene ring or a cyclohexane ring;

[0226] X 2 Each independently is -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)-(CH2) q - (p and q are each independently an integer from 0 to 3);

[0227] X 3 is a single bond, -(CH2) r -O-, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O- or -(CH2) r -OC(=O)-(r is an integer from 0 to 3);

[0228] R 2 is a hydrogen atom, an optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 6-14 aryl, optionally substituted 3-8 membered monocyclic non-aromatic heterocyclic group or tri(C 1-6 Alkyl)silyl;

[0229] n is an integer from 1 to 3;

[0230] m is an integer from 1 to 3; and

[0231] When m is 1, m' is 0 or 1; when m is 2 or 3, m' is 1.

[0232] In this embodiment, more preferably R 2 is a hydrogen atom, an optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl or optionally substituted C 6-14 Aryl.

[0233] More preferably, compound (I) is a compound wherein:

[0234] R 1 Each independently is a straight chain C 12-28 alkyl;

[0235] X 1 are each independently -O-, -C(=O)O- or -OC(=O)-;

[0236] Ring A 2 and Ring B 1 Each is independently a benzene ring or a cyclohexane ring;

[0237] X 2 Each independently is -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)-(CH2) q - (p and q are each independently an integer from 0 to 3);

[0238] X 3is a single bond, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O- or -(CH2) r -OC(=O)-(r is an integer from 0 to 3);

[0239] R 2 is a hydrogen atom, an optionally substituted C 1-6 Alkyl or 3-8 membered monocyclic non-aromatic heterocyclic group or tri(C 1-6 Alkyl)silyl;

[0240] n is an integer from 1 to 3;

[0241] m is an integer from 1 to 3; and

[0242] When m is 1, m' is 0 or 1; when m is 2 or 3, m' is 1.

[0243] In this embodiment, more preferably R 2 is a hydrogen atom or an optionally substituted C 1-6 alkyl.

[0244] Still more preferred compounds (I) are compounds wherein:

[0245] R 1 Each independently is a straight chain C 12-28 alkyl;

[0246] X 1 are each independently -O-, -C(=O)O- or -OC(=O)-;

[0247] Ring A 2 and Ring B 1 Each is independently a benzene ring or a cyclohexane ring;

[0248] X 2 Each independently is -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)-(CH2) q - (p and q are each independently an integer from 0 to 3);

[0249] X 3 is a single bond, -(CH2) r -C(=O)-, -(CH2)r -C(=O)O- or -(CH2) r -OC(=O)-(r is an integer from 0 to 3);

[0250] R 2 is a hydrogen atom, optionally replaced by C 1-6 Alkanoylamino (eg, acetylamino) substituted C 1-6 alkyl or a 3-8 membered monocyclic non-aromatic heterocyclic group (e.g., morpholin-4-yl) or a tri(C 1-6 alkyl)silyl (e.g., triisopropylsilyl);

[0251] n is an integer from 1 to 3;

[0252] m is an integer from 1 to 3; and

[0253] When m is 1, m' is 0 or 1; when m is 2 or 3, m' is 1.

[0254] Further preferred compounds (I) are compounds wherein:

[0255] R 1 Each independently is a straight chain C 14-26 alkyl;

[0256] X 1 for -O-;

[0257] Ring A 2 and Ring B 1 Each is independently a benzene ring or a cyclohexane ring;

[0258] X 2 Each is independently a single bond, -(CH2) p -O-(CH2) q -, -C(=O)-, -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)- (p and q are each independently an integer of 0 or 1);

[0259] X 3 is a single bond, -C(=O)-, -C(=O)O- or -OC(=O)-;

[0260] R 2 is a hydrogen atom, optionally replaced by C 1-6 Alkanoylamino (eg, acetylamino) substituted C 1-6 alkyl or a 3-8 membered monocyclic non-aromatic heterocyclic group (e.g., morpholin-4-yl) or a tri(C 1-6 alkyl)silyl (e.g., triisopropylsilyl);

[0261] n is an integer from 1 to 3;

[0262] m is 1 or 3; and

[0263] When m is 1, m' is 0 or 1; when m is 3, m' is 1.

[0264] Among the above further preferred compounds (I), R 2 A hydrogen atom or C 1-6 alkyl.

[0265] In the above further preferred compound (I), when m is 1 and m' is 0, X 2 Each is independently a single bond, -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)- (p and q are each independently an integer of 0 or 1), R 2 C 1-6 alkyl.

[0266] In the above further preferred compound (I), when m is 1 or 3 and m' is 1, X 2 Each independently is -(CH2) p -O-(CH2) q - or -C(=O)- (p and q are each independently an integer of 0 or 1), X 3 is a single bond or -C(=O)O-, and R 2 A hydrogen atom or C 1-6 alkyl.

[0267] Particularly preferred compounds (I) are compounds wherein:

[0268] R 1 Each independently is a straight chain C 14-26 alkyl;

[0269] X 1 for -O-;

[0270] Ring A 2 and Ring B 1 Each is a benzene ring;

[0271] X 2 Each is independently a single bond, -(CH2) p -O-(CH2) q -, -C(=O)-, -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)- (p and q are each independently an integer of 0 or 1);

[0272] X3 is a single bond, -C(=O)-, -C(=O)O- or -OC(=O)-;

[0273] R 2 A hydrogen atom or C 1-6 alkyl;

[0274] n is an integer from 1 to 3;

[0275] m is 1 or 3; and

[0276] When m is 1, m' is 0 or 1; when m is 3, m' is 1.

[0277] In the above particularly preferred compounds (I), more preferably when m is 1 and m' is 0, X 2 is a single bond, -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)- (p and q are each independently an integer of 0 or 1), and R 2 C 1-6 alkyl.

[0278] In the above particularly preferred compounds (I), more preferably when m is 1 or 3 and m' is 1, X 2 -(CH2) p -O-(CH2) q - or -C(=O)- (p and q are each independently an integer of 0 or 1), and X 3 It is a single bond or -C(=O)O-.

[0279] Preferred compounds (II) are those wherein:

[0280] R 1 Each independently is a straight chain C 10-30 alkyl;

[0281] X 1 are each independently a single bond, -O-, -C(=O)-, -C(=O)O- or -OC(=O)-;

[0282] Ring A 2 and Ring B 1 Each is independently a benzene ring or a cyclohexane ring;

[0283] X 2 Each independently is -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2)p -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)-(CH2) q - (p and q are each independently an integer from 0 to 3);

[0284] X 3 is a single bond, -(CH2) r -O-, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O- or -(CH2) r -OC(=O)-(r is an integer from 0 to 3);

[0285] R 2 is a hydrogen atom, an optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 6-14 aryl, optionally substituted 3-8 membered monocyclic non-aromatic heterocyclic group or tri(C 1-6 Alkyl)silyl;

[0286] m is an integer from 1 to 3; and

[0287] When m is 1, m' is 0 or 1; when m is 2 or 3, m' is 1.

[0288] In this embodiment, more preferably R 2 is a hydrogen atom, an optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl or optionally substituted C 6-14 Aryl.

[0289] More preferred compounds (II) are compounds wherein:

[0290] R 1 Each independently is a straight chain C 12-28 alkyl;

[0291] X 1 are each independently -O-, -C(=O)O- or -OC(=O)-;

[0292] Ring A 2 and Ring B 1 Each is independently a benzene ring or a cyclohexane ring;

[0293] X 2 Each independently is -(CH2) p -、-(CH2)p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)-(CH2) q - (p and q are each independently an integer from 0 to 3);

[0294] X 3 is a single bond, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O- or -(CH2) r -OC(=O)-(r is an integer from 0 to 3);

[0295] R 2 is a hydrogen atom, an optionally substituted C 1-6 Alkyl or 3-8 membered monocyclic non-aromatic heterocyclic group or tri(C 1-6 Alkyl)silyl;

[0296] m is an integer from 1 to 3; and

[0297] When m is 1, m' is 0 or 1; when m is 2 or 3, m' is 1.

[0298] In this embodiment, more preferably R 2 is a hydrogen atom or an optionally substituted C 1-6 alkyl.

[0299] Still more preferred compounds (II) are compounds wherein:

[0300] R 1 Each independently is a straight chain C 12-28 alkyl;

[0301] X 1 are each independently -O-, -C(=O)O- or -OC(=O)-;

[0302] Ring A 2 and Ring B 1 Each is independently a benzene ring or a cyclohexane ring;

[0303] X 2 Each independently is -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2)q -、-(CH2) p -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)-(CH2) q - (p and q are each independently an integer from 0 to 3);

[0304] X 3 is a single bond, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O- or -(CH2) r -OC(=O)-(r is an integer from 0 to 3);

[0305] R 2 is a hydrogen atom, optionally replaced by C 1-6 Alkanoylamino (e.g., acetylamino) substituted C 1-6 alkyl or a 3-8 membered monocyclic non-aromatic heterocyclic group (e.g., morpholin-4-yl) or a tri(C 1-6 alkyl)silyl (e.g., triisopropylsilyl);

[0306] m is an integer from 1 to 3; and

[0307] When m is 1, m' is 0 or 1; when m is 2 or 3, m' is 1.

[0308] Further preferred compounds (II) are compounds wherein:

[0309] R 1 Each independently is a straight chain C 14-26 alkyl;

[0310] X 1 for -O-;

[0311] Ring A 2 and Ring B 1 Each is a benzene ring or a cyclohexane ring;

[0312] X 2 Each is independently a single bond, -(CH2) p -O-(CH2) q -, -C(=O)-, -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)- (p and q are each independently an integer of 0 or 1);

[0313] X 3 is a single bond, -C(=O)-, -C(=O)O- or -OC(=O)-;

[0314] R 2 is a hydrogen atom, optionally replaced by C 1-6 Alkanoylamino (e.g., acetylamino) substituted C 1-6 alkyl or a 3-8 membered monocyclic non-aromatic heterocyclic group (e.g., morpholin-4-yl) or a tri(C 1-6 alkyl)silyl (e.g., triisopropylsilyl);

[0315] m is 1 or 3; and

[0316] When m is 1, m' is 0 or 1; when m is 3, m' is 1.

[0317] Among the above further preferred compounds (II), R 2 A hydrogen atom or C 1-6 alkyl.

[0318] In the above further preferred compound (II), when m is 1 and m' is 0, X 2 Each is independently a single bond, -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)- (p and q are each independently an integer of 0 or 1), and R 2 C 1-6 alkyl.

[0319] In the above further preferred compound (II), when m is 1 or 3 and m' is 1, X 2 Each independently is -(CH2) p -O-(CH2) q - or -C(=O)- (p and q are each independently an integer of 0 or 1), X 3 is a single bond or -C(=O)O-, and R 2 A hydrogen atom or C 1-6 alkyl.

[0320] Particularly preferred compounds (II) are those wherein:

[0321] R 1 Each independently is a straight chain C 14-26 alkyl;

[0322] X 1 for -O-;

[0323] Ring A 2 and Ring B 1 Each is a benzene ring;

[0324] X 2 Each is independently a single bond, -(CH2) p -O-(CH2)q -, -C(=O)-, -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)- (p and q are each independently an integer of 0 or 1);

[0325] X 3 is a single bond, -C(=O)-, -C(=O)O- or -OC(=O)-;

[0326] R 2 A hydrogen atom or C 1-6 alkyl;

[0327] m is 1 or 3; and

[0328] When m is 1, m' is 0 or 1; when m is 3, m' is 1.

[0329] In the above particularly preferred compounds (II), more preferably when m is 1 and m' is 0, X 2 is a single bond, -C(=O)O-(CH2) q -or-(CH2) p -OC(=O)- (p and q are each independently an integer of 0 or 1), and R 2 C 1-6 alkyl.

[0330] In the above particularly preferred compounds (II), more preferably when m is 1 or 3 and m' is 1, X 2 -(CH2) p -O-(CH2) q - or -C(=O)- (p and q are each independently an integer of 0 or 1), and X 3 It is a single bond or -C(=O)O-.

[0331] Specific examples included in the above-mentioned compound (I) include compounds represented by the following formulas (IV-a) to (IV-n). Among the specific examples, preferred are compound (IV-a) to compound (IV-l), more preferred are compound (IV-a) to compound (IV-j), further preferred are compound (IV-a) to compound (IV-g), particularly preferred are compound (IV-a) to compound (IV-e), and most preferred are compound (IV-a) and compound (IV-b).

[0332]

[0333]

[0334] Another embodiment of the precipitation promoter of the present invention is a group having one or more linear C 10-40 alkyl, and further optionally substituted C 1-10 Alkanes (hereinafter sometimes referred to as "alkane compounds").

[0335] C 1-10 Alkanes can be straight or branched chains. 1-10 The carbon number of the alkane is preferably 1-8, more preferably 2-6, and further preferably 3-6.

[0336] Alkanes have straight-chain C 10-40 The alkyl group is preferably a straight chain C 10-30 Alkyl, more preferably straight chain C 12-28 Alkyl, more preferably a straight chain C 14-26 Alkyl. Alkyl compounds have a straight chain C 10-40 The number of the alkyl groups is preferably 2-6, more preferably 2-4, further preferably 3 or 4.

[0337] The straight chain C 10-40 Alkyl and C 1-10 The alkane-bonded group is preferably a group selected from -O-, -C(=O)-, -C(=O)O- and -OC(=O)-, and more preferably -O-.

[0338] Preferred alkane compounds are those having 2-6 straight-chain C 10-30 Alkyl and optionally substituted C 1-8 Alkanes. More preferred alkane compounds are straight-chain C 10-30 Alkyl and optionally substituted C 2-6 Alkanes. Further preferred alkane compounds are those having 3 or 4 straight-chain C 14-26 Alkyl and optionally substituted C 3-6 Alkane.

[0339] Specific examples of the alkane compound include compounds represented by the following formula (Va) or (Vb):

[0340]

[0341] [Preparation method of the precipitation accelerator of the present invention]

[0342] For example, where X 1The compound (I) wherein m is -O-, m is 1 and m' is 0 can be prepared by using a halogen atom (X a ) is prepared by alkylating the hydroxyl group of compound (IA-2). Alkylation is well known in the field of organic synthesis, and a person skilled in the art can perform alkylation by appropriately setting the conditions for the alkylation.

[0343]

[0344] Where X a is a halogen atom (preferably a chloride ion or a bromide ion), and the other symbols are as defined above.

[0345] For example, where X 2 The compound (I) wherein m' is 1 and is -CH2-O- can be prepared by using a halogen atom (X a ) can be prepared by alkylating the hydroxyl group of compound (IB).

[0346]

[0347] The symbols are as defined above.

[0348] Other precipitation promoters of the present invention can also be prepared from starting compounds according to methods known per se (e.g., alkylation, esterification, amidation, etc.) or methods analogous thereto. As starting compounds, commercially available products can be used, or compounds prepared according to methods known per se or methods analogous thereto can be used.

[0349] [Pseudo solid phase protecting group of the present invention]

[0350] The precipitation accelerator of the present invention is used to precipitate an organic compound protected by an organic group having one or more aliphatic hydrocarbon groups having 10 or more carbon atoms (i.e., the pseudo solid phase protecting group of the present invention) from a solvent containing the compound. Specific examples of the pseudo solid phase protecting group of the present invention and the organic compound protected by the pseudo solid phase protecting group include nucleosides, nucleotides, etc. disclosed in WO2012 / 157723, WO 2012 / 157723, WO 2014 / 189142, JP-A-2010-116418, etc., and amino acids, peptides, etc. disclosed in WO 2010 / 104169, WO 2010 / 113939, WO 2011 / 078295, WO 2012 / 029794, JP-A-2009-185063, JP-A-2010-275254, etc. The pseudo solid phase protecting group of the present invention is preferably an organic group which is not removed under acidic conditions but can be removed under basic conditions.

[0351] One embodiment of the pseudo solid phase protecting group of the present invention is, for example, 6-14 Aromatic hydrocarbon ring organic group, the C 6-14 The aromatic hydrocarbon ring is bonded to the "hydrocarbon group to which an aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group" via a linking group. As the above-mentioned hydrocarbon group, those described above can be repeated, and an aliphatic hydrocarbon group, an aromatic aliphatic hydrocarbon group (e.g., a benzyl group), and a group in which a monocyclic saturated hydrocarbon group is bonded to an aliphatic hydrocarbon group (e.g., a cyclohexylmethyl group) are preferred. The "hydrocarbon group to which an aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group" also includes the "aliphatic hydrocarbon group having 10 or more carbon atoms" itself (i.e., the "aliphatic hydrocarbon group to which an aliphatic hydrocarbon group is bonded via a single bond").

[0352] Although the aliphatic hydrocarbon group having 10 or more carbon atoms in the pseudo solid phase protecting group of the present invention may be straight chain or branched, it is preferably straight chain in order to fully exert the effect of the precipitation accelerator of the present invention. The aliphatic hydrocarbon group having 10 or more carbon atoms is more preferably selected from straight chain C 10-40 Alkyl and straight chain C 10-40 Alkenyl groups, more preferably straight chain C 10-40 Alkyl, more preferably a straight chain C 10-30 Alkyl, particularly preferably straight chain C 12-28 Alkyl, and most preferably a straight chain C 14-26 alkyl.

[0353] The above-mentioned linking group is preferably selected from -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH-, -NHC(=O)-, -S-, -SO-, -SO2-, -Si(R)(R')O-, -Si(R)(R')- (R, R' are each independently a hydrogen atom or C 1-22 The hydrocarbon group is more preferably selected from -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- and -NHC(=O)-, and more preferably -O-.

[0354] C 6-14 The aromatic hydrocarbon ring is preferably selected from a benzene ring and a naphthalene ring, and is more preferably a benzene ring.

[0355] The above “C 6-14 The aromatic hydrocarbon ring is bonded to a hydrocarbon group having 10 or more carbon atoms bonded to an aliphatic hydrocarbon group via a single bond or a linking group via a linking group, preferably a C 6-14 An aromatic hydrocarbon ring is bonded to a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms via a linking group", more preferably "C 6-14 An aromatic hydrocarbon ring, which is connected to a linear C 10-40 Alkyl and straight chain C 10-40 More preferably, it is a "C6-14 Aromatic hydrocarbon ring, which is connected to the straight chain C 10-40 Alkyl bond", more preferably "C 6-14 Aromatic hydrocarbon ring, which is connected to the straight chain C 10-30 Alkyl bond", particularly preferably "C 6-14 Aromatic hydrocarbon ring, which is connected to the straight chain C 12-28 Alkyl bonded", and most preferably "C 6-14 Aromatic hydrocarbon ring, which is connected to the straight chain C 14-26 In these groups, the linking group is preferably -O-. In these groups, the C 6-14 The aromatic hydrocarbon ring is preferably a benzene ring.

[0356] The pseudo solid phase protecting group is preferably an organic group having a benzene ring, wherein the benzene ring is bonded to a straight-chain C 10-40 The term "alkyl" is more preferably "an organic group having a benzene ring, the benzene ring being connected to a straight-chain C 10-40 Alkyl bond", further preferably "an organic group having a benzene ring, the benzene ring is connected to a straight chain C 10-30 "alkyl bond", particularly preferably "an organic group having a benzene ring, the benzene ring is connected to a straight-chain C 12-28 Alkyl bond", the most preferred is "an organic group having a benzene ring, the benzene ring is connected to a straight chain C 14-26 Alkyl bonding".

[0357] The pseudo solid phase protecting group of the present invention is, for example, a group represented by the following formula (III) (hereinafter referred to as "pseudo solid phase protecting group (III)"):

[0358] **LYZ (III)

[0359] In the formula,

[0360] ** Indicates the bonding position to the protected group;

[0361] L represents a single bond, or a group represented by formula (a1) or (a1'):

[0362]

[0363] In the formula,

[0364] * represents the bonding position with Y;

[0365] ** Same as the above definition;

[0366] R 8 and R9 Each independently represents C 1-22 Hydrocarbon;

[0367] L1 indicates divalent C 1-22 a hydrocarbon group; and

[0368] L2 represents a single bond, or ** C(=O)N(R 2’ )-R 1’ -N(R 3 ) *** The group shown in the formula ** represents the bonding position with L1, *** Indicates the bonding position with C=O, R 1’ Represents C 1-22 Alkylene, R 2’ and R 3 Each independently represents a hydrogen atom or a C 1-22 Alkyl, or R 2’ and R 3 optionally forming a ring together;

[0369] Y represents a single bond, an oxygen atom or NR, wherein R represents a hydrogen atom, an alkyl group or an aralkyl group; and

[0370] Z represents a group represented by formula (a2), formula (a2') or formula (a2") {preferably formula (a2) or formula (a2')}:

[0371]

[0372] In the formula,

[0373] * Indicates the bonding position;

[0374] R 4 is a hydrogen atom, or when R b When it is a group represented by the following formula (a3), R 4 Optionally with R of ring C 6 Together they represent a single bond or -O-, and together with Ring A or Ring B and Ring C form a condensed ring (preferably a fluorene ring or a xanthene ring);

[0375] k Qs each independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0376] k R 5 each independently represents a hydrocarbon group to which an aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group (preferably a hydrocarbon group to which a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group);

[0377] k represents an integer from 1 to 4;

[0378] Ring A and Ring B each independently have k QR 5 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C optionally substituted by halogen atoms 1-6 Alkoxy;

[0379] R a represents a hydrogen atom, or a phenyl group optionally substituted by a halogen atom (preferably a hydrogen atom); and

[0380] R b represents a hydrogen atom, or a group represented by formula (a3):

[0381]

[0382] In the formula, * Indicates the bonding position;

[0383] j represents an integer from 0 to 4;

[0384] j Qs each independently represent the same meaning as above;

[0385] j R 7 each independently represents a hydrocarbon group to which an aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group (preferably a hydrocarbon group to which a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group);

[0386] R 6 represents a hydrogen atom, or optionally connected to R of ring A or ring B 4 Together they represent a single bond or -O-, and together with Ring A or Ring B and Ring C form a condensed ring (preferably a fluorene ring or a xanthene ring); and

[0387] In addition to having j QR 7 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C optionally substituted by halogen atoms 1-6 Alkoxy, or

[0388] R a and R b together to form a carbonyl group.

[0389] Examples of the group protected by the pseudo solid phase protecting group (III) include hydroxyl, amino, carboxyl.

[0390] R in formula (a2), formula (a2') and formula (a2") 5, and R in formula (a3) 7 The aliphatic hydrocarbon groups having 10 or more carbon atoms are each independently selected from linear C 10-40 Alkyl and straight chain C 10-40 Alkenyl groups, more preferably straight chain C 10-40 Alkyl, more preferably a straight chain C 10-30 Alkyl, particularly preferably straight chain C 12-28 Alkyl, most preferably straight chain C 14-26 alkyl.

[0391] R in formula (a2), formula (a2') and formula (a2") 5 , and R in formula (a3) 7 The linking groups possessed are each independently preferably -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-, and more preferably -O-.

[0392] R in formula (a2), formula (a2') and formula (a2") 5 , and R in formula (a3) 7 (i.e., a hydrocarbon group to which an aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group) is each independently preferably selected from a linear C 10-40 Alkyl and straight chain C 10-40 Alkenyl groups, more preferably straight chain C 10-40 Alkyl, more preferably a straight chain C 10-30 Alkyl, particularly preferably straight chain C 12-28 Alkyl, most preferably straight chain C 14-26 alkyl.

[0393] In the formula (a2), the formula (a2'), the formula (a2") and the formula (a3), Q is preferably -O-.

[0394] In formula (III), a preferred embodiment of L represented by formula (a1) is a group wherein:

[0395] L1 is divalent C 1-22 Hydrocarbyl or CH2-O-1,4-phenylene-O-CH2; and

[0396] L2 is a single bond, or ** C(=O)N(R 2’ )-R 1’ -N(R 3 ) *** The groups shown, where ** represents the bonding position with L1, *** Indicates the bonding position with C=O, R 1’ C 1-6Alkylene, R 2’ and R 3 are each independently a hydrogen atom or an optionally substituted C 1-6 Alkyl, or R 2’ and R 3 Optionally together form an optionally substituted C 1-6 Alkylene bond.

[0397] Another preferred embodiment of L represented by formula (a1) is a group wherein:

[0398] L1 is divalent C 1-22 a hydrocarbon group; and

[0399] L2 is a single bond.

[0400] Another preferred embodiment of L represented by formula (a1) is a group wherein:

[0401] L1 is ethylene; and

[0402] L2 is ** C(=O)N(R 2’ )-R 1’ -N(R 3 ) *** The groups shown, where ** represents the bonding position with L1, *** Indicates the bonding position with C=O, R 1’ C 1-22 Alkylene, R 2’ and R 3 are each independently a hydrogen atom or a C 1-22 Alkyl, or R 2’ and R 3 Optionally forming a ring together.

[0403] Another preferred embodiment of L represented by formula (a1) is a group wherein:

[0404] L1 is ethylene; and

[0405] L2 is ** C(=O)N(R 2’ )-R 1’ -N(R 3 ) *** The groups shown, where ** represents the bonding position with L1, *** indicates the bonding position to C=O, and N(R 2’ )-R 1’ -N(R 3 ) portion to form a piperazinediyl group (e.g., 1,4-piperazinediyl).

[0406] Another preferred embodiment of L represented by formula (a1) is a group wherein:

[0407] L1 is ethylene; and

[0408] L2 is ** C(=O)N(R 2’ )-R 1’ -N(R 3 ) *** The groups shown, where ** represents the bonding position with L1, *** Indicates the bonding position with C=O, R 1’ is pentylene or hexylene, and R 2’ and R 3 Each is independently a hydrogen atom or a methyl group.

[0409] A particularly preferred embodiment of L represented by formula (a1) is a succinyl group, which is easily available at low cost.

[0410] Now, L represented by the formula (a1′) in the formula (III) is explained.

[0411] L1 in formula (a1') is preferably a divalent C 6-10 The aromatic hydrocarbon group is more preferably a phenylene group.

[0412] L2 in formula (a1') is preferably a single bond.

[0413] The preferred combination of L1 and L2 in formula (a1') is that L1 is a divalent C 6-10 A combination in which the aromatic hydrocarbon group and L2 are a single bond. A more preferred combination of L1 and L2 in formula (a1′) is a combination in which L1 is a phenylene group and L2 is a single bond.

[0414] In formula (a1'), R 8 and R 9 Each is independently preferably C 1-22 Alkyl, more preferably C 1-10 alkyl.

[0415] A preferred embodiment of L represented by formula (a1') is a group wherein:

[0416] R 8 and R 9 Each independently is C 1-22 alkyl;

[0417] L1 is divalent C 6-10 an aromatic hydrocarbon group; and

[0418] L2 is a single bond.

[0419] Another preferred embodiment of L represented by formula (a1') is a group wherein:

[0420] R 8 and R 9 Each independently is C 1-10 alkyl;

[0421] L1 is phenylene; and

[0422] L2 is a single bond.

[0423] In formula (III), Y is a single bond, an oxygen atom or NR (R is a hydrogen atom, an alkyl group or an aralkyl group). When Y is a single bond, L is a single bond, Z is a group represented by formula (a2), and R 4 is a hydrogen atom, and R a and R b Preferably, they are formed together with a carbonyl group.

[0424] In formula (III), Y is preferably an oxygen atom or NR (R is a hydrogen atom, an alkyl group or an aralkyl group), and R is preferably a hydrogen atom, C 1-6 Alkyl or C 7-16 The aralkyl group is more preferably a hydrogen atom, a methyl group, an ethyl group or a benzyl group, and particularly preferably a hydrogen atom. Y is particularly preferably an oxygen atom.

[0425] Z in formula (III) is preferably a group represented by formula (a2) or formula (a2'), and more preferably a group represented by formula (a2). 4 Preferably, it is a hydrogen atom. a and R b Each is preferably a hydrogen atom.

[0426] A preferred embodiment of Z represented by formula (a2) is a group wherein:

[0427] R a and R b Each is a hydrogen atom;

[0428] R 4 is a hydrogen atom;

[0429] k is an integer from 1 to 3;

[0430] k Qs are each -O-; and

[0431] k R 5 Each independently is a straight chain C 10-40 alkyl.

[0432] When Z is this embodiment, it is more preferred that L is a succinyl group, or a group represented by formula (a1') (in formula (a1'), R 8 and R 9Each independently is C 1-10 alkyl, L1 is a divalent phenylene group, L2 is a single bond), and Y is an oxygen atom, or LY is a single bond, and more preferably L is a succinyl group, or a group represented by formula (a1') (in formula (a1'), R 8 and R 9 Each independently is C 1-10 alkyl, L1 is a divalent phenylene group, and L2 is a single bond), and Y is an oxygen atom.

[0433] Another preferred embodiment of Z represented by formula (a2) is a group wherein:

[0434] R a and R b Each is a hydrogen atom;

[0435] R 4 is a hydrogen atom;

[0436] k is an integer from 1 to 3;

[0437] k Qs are each -O-; and

[0438] k R 5 Each independently has 1 to 3 straight-chain C 10-40 Benzyl, or 1 to 3 straight-chain C 10-40 alkyl cyclohexylmethyl; and

[0439] In addition to k QRs, ring A 5 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C optionally substituted by halogen atoms 1-6 Alkoxy.

[0440] When Z is this embodiment, it is more preferred that L is a succinyl group or a group represented by formula (a1') (in formula (a1'), R 8 and R 9 Each independently is C 1-10 alkyl, L1 is a divalent phenylene group, and L2 is a single bond), and Y is an oxygen atom, or LY is a single bond, and further preferably L is a succinyl group or a group represented by formula (a1') (in formula (a1'), R 8 and R 9 Each independently is C 1-10 alkyl, L1 is a divalent phenylene group, and L2 is a single bond), and Y is an oxygen atom.

[0441] Another preferred embodiment of Z represented by formula (a2) is a group wherein:

[0442] Ra and R 4 each a hydrogen atom; and

[0443] R b is a group represented by formula (a3), wherein * is the bonding position, j is an integer from 0 to 3, j Qs are -O-, j R 7 Each independently is C 10-40 Alkyl, R 6 A hydrogen atom.

[0444] When Z is this embodiment, it is more preferred that L is a succinyl group or a group represented by formula (a1') (in formula (a1'), R 8 and R 9 Each independently is C 1-10 alkyl, L1 is a divalent phenylene group, and L2 is a single bond), and Y is an oxygen atom, or LY is a single bond, and further preferably L is a succinyl group or a group represented by formula (a1') (in formula (a1'), R 8 and R 9 Each independently is C 1-10 alkyl, L1 is a divalent phenylene group, and L2 is a single bond), and Y is an oxygen atom.

[0445] Still another preferred embodiment of Z represented by formula (a2) is a group wherein:

[0446] R a is a hydrogen atom; and

[0447] R b is a group represented by formula (a3), wherein * is the bonding position, j is an integer from 0 to 3, j Qs are -O-, j R 7 Each independently is a straight chain C 10-40 Alkyl, and R 6 R 4 Together, they form a single bond or -O-, whereby Ring A and Ring C together form a fluorene ring or a xanthene ring.

[0448] When Z is this embodiment, it is more preferred that L is a succinyl group or a group represented by formula (a1') (in formula (a1'), R 8 and R 9 Each independently is C 1-10 alkyl, L1 is a divalent phenylene group, and L2 is a single bond), and Y is an oxygen atom, or LY is a single bond, and further preferably L is a succinyl group or a group represented by formula (a1') (in formula (a1'), R 8 and R 9 Each independently is C 1-10alkyl, L1 is a divalent phenylene group, and L2 is a single bond), and Y is an oxygen atom.

[0449] Another preferred embodiment of Z represented by formula (a2) is a group wherein:

[0450] R a and R b Together they form a carbonyl group;

[0451] R 4 is a hydrogen atom;

[0452] k is an integer from 1 to 3;

[0453] k Qs are -O-; and

[0454] k R 5 Each independently is a straight chain C 10-40 alkyl.

[0455] When Z is this embodiment, it is more preferred that LY is a single bond or succinyl-1,4-piperazinediyl, and further preferred that LY is a single bond.

[0456] Another preferred embodiment of Z represented by formula (a2) is a group wherein:

[0457] R a and R b Together they form a carbonyl group;

[0458] R 4 is a hydrogen atom;

[0459] k is an integer from 1 to 3;

[0460] k Qs are each -O-; and

[0461] k R 5 Each independently has 1 to 3 straight-chain C 10-40 Benzyl, or 1 to 3 straight-chain C 10-40 alkyl cyclohexylmethyl; and

[0462] In addition to k QRs, ring A 5 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C optionally substituted by halogen atoms 1-6 Alkoxy.

[0463] When Z is this embodiment, it is more preferred that LY is a single bond or succinyl-1,4-piperazinediyl, and further preferred that LY is a single bond.

[0464] The pseudo solid phase protecting group (III) is preferably a group that is not removed under acidic conditions but can be removed under basic conditions.

[0465] Specific examples 1 of the pseudo solid phase protecting group (III) having a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms (for protecting a hydroxyl group) include

[0466] Such a group, in which:

[0467] L is a succinyl group or a group represented by formula (a1') (in formula (a1'), R 8 and R 9 Each independently is C 1-10 Alkyl, L 1 is a divalent phenylene group, and L 2is a single bond), and YZ is 3,4,5-trioctadecyloxybenzyloxy, 2,4-dioctadecyloxybenzyloxy, 2,4-didocosyloxybenzyloxy, 3,5-didocosyloxybenzyloxy, 3,5-bis[3',4',5'-trioctadecyloxybenzyloxy]benzyloxy, 3,4,5-tri[3',4',5'-trioctadecyloxybenzyloxy]benzyloxy, 3,4,5-trioctadecyloxybenzylamino, 2,4-didocosyloxybenzylamino, 3,5-didocosyloxybenzylamino, bis(4-docosyloxyphenyl)methylamino , 4-methoxy-2-[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino, 4-methoxy-2-[3',4',5'-tri(octadecyloxy)cyclohexylmethoxy]benzylamino, 2,4-bis(dodecyloxy)benzylamino, phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino, bis[4-(12-docosyloxydodecyloxy)phenyl]methylamino, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino, 3,4,5-tris[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino, 3,4, 5-Tris(octadecyloxy)cyclohexylmethoxy, 3,5-di(docosyloxy)cyclohexylmethoxy, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethoxy, 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethoxy, 3,4,5-tris(octadecyloxy)cyclohexylmethylamino, 2,4-di(docosyloxy)cyclohexylmethylamino, 3,5-di(docosyloxy)cyclohexylmethylamino, bis(4-docosyloxyphenyl)methylamino, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethoxy 4-(octadecyloxy)cyclohexylmethoxy)cyclohexylmethylamino, 4-methoxy-2-[3',4',5'-tri(octadecyloxy)cyclohexylmethoxy]cyclohexylmethylamino, 2,4-bis(dodecyloxy)cyclohexylmethylamino, phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino, bis[4-(12-docosyloxydodecyloxy)phenyl]methylamino, 3,5-bis[3',4',5'-tri(octadecyloxy)cyclohexylmethoxy]cyclohexylmethylamino, or 3,4,5-tris[3',4',5'-tri(octadecyloxy)cyclohexylmethoxy]cyclohexylmethylamino;

[0468] Such a group, in which:

[0469] LY is succinyl-1,4-piperazinediyl, and Z is 3,4,5-tri(octadecyloxy)benzoyl, 2,4-di(octadecyloxy)benzoyl, 2,4-di(docosyloxy)benzoyl, 3,5-di(docosyloxy)benzoyl, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzoyl, or 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzoyl; and

[0470] Such a group, in which:

[0471] LY is a single bond, and Z is a (3,5-bisdocosyloxyphenyl)diphenylmethyl group or a bis(4-chlorophenyl)(3,5-bisdocosyloxyphenyl)methyl group.

[0472] Specific examples 2 of the pseudo solid phase protecting group (III) having a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms (which is used to protect an amino group or an imino group) include a group wherein LY is a single bond, and Z is 3,4,5-tri(octadecyloxy)benzoyl, 2,4-di(octadecyloxy)benzoyl, 2,4-di(docosyloxy)benzoyl, 3,5-di(docosyloxy)benzoyl, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzoyl, 3,4,5-tris[3',4',5'-tri(octadecyloxy)benzyloxy]benzoyl, (3,5-didocosyloxyphenyl)diphenylmethyl, or bis(4-chlorophenyl)(3,5-didocosyloxyphenyl)methyl.

[0473] Specific examples 3 of the pseudo solid phase protecting group (III) having a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms (which is used to protect a carboxyl group) include groups wherein LY is a single bond, and Z is 3,4,5-tri(octadecyloxy)benzyl, 2,4-di(octadecyloxy)benzyl, 2,4-di(docosyloxy)benzyl, 3,5-di(docosyloxy)benzyl, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzyl, 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzyl, (3,5-didocosyloxyphenyl)diphenylmethyl, or bis(4-chlorophenyl)(3,5-didocosyloxyphenyl)methyl.

[0474] Specific examples 4 of the pseudo solid phase protecting group (III) having a branched aliphatic hydrocarbon group having 10 or more carbon atoms (for protecting a hydroxyl group) include a group wherein L is a succinyl group or a group represented by formula (a1′) (in formula (a1′), R 8 and R 9 Each independently is C 1-10 Alkyl, L 1is a divalent phenylene group, and L 2 is a single bond), and YZ is 4-(2',3'-dihydrophytyloxy)benzyloxy, 4-(3,7,11-trimethyldodecyloxy)benzyloxy, 4-[2,2,4,8,10,10-hexamethyl-5-dodecylamino]benzyloxy, 4-(2',3'-dihydrophytyloxy)-2-methylbenzyloxy, 4-(2',3'-dihydrophytyloxy)-2-methoxybenzyloxy, 2,4-bis(2',3'-dihydrophytyloxy)benzyloxy, 3,5-bis(2',3'- benzyloxy, 2-[3',4',5'-tris(2",3'-dihydrophytyloxy)benzyloxy]-4-methoxybenzyloxy, 3,4,5-tris(2',3'-dihydrophytyloxy)benzyloxy, 3,4,5-tris[3,4,5-tris(2',3'-dihydrophytyloxy)benzyloxy]benzyloxy, 2,4-di(2-decyltetradecyloxy)benzyloxy, 4-(2',3'-dihydrophytyloxy)benzylamino, 4-(3,7,11-trimethyldodecyloxy )benzylamino, 4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylamino, 4-(2',3'-dihydrophytyloxy)-2-methylbenzylamino, 4-(2',3'-dihydrophytyloxy)-2-methoxybenzylamino, 2,4-bis(2',3'-dihydrophytyloxy)benzylamino, 3,5-bis(2',3'-dihydrophytyloxy)benzylamino, 2-[3',4',5'-tris(2",3"-dihydrophytyloxy)benzyloxy] -4-methoxybenzylamino, 3,4,5-tris(2',3'-dihydrophytyloxy)benzylamino, 3,4,5-tris[3,4,5-tris(2',3'-dihydrophytyloxy)benzyloxy]benzylamino, {[(2-chloro-5-(2',3'-dihydrophytyloxy)phenyl)]phenylmethyl}amino, {[2,3,4-tris(2',3'-dihydrophytyloxy)phenyl]phenylmethyl}amino, or {bis[4-(2',3'-dihydrophytyloxy)phenyl]methyl}amino.

[0475] Here, 2,3-dihydrophytyl refers to 3,7,11,15-tetramethylhexadecyl. The same applies to 2',3'-dihydrophytyl and 2",3"-dihydrophytyl.

[0476] Specific examples 5 of the pseudo solid phase protecting group (III) having a branched aliphatic hydrocarbon group having 10 or more carbon atoms (which is used to protect an amino group or an imino group) include groups wherein LY is a single bond, and Z is 4-(2',3'-dihydrophytyloxy)benzoyl, 4-(3,7,11-trimethyldodecyloxy)benzoyl, 4-[2,2,4,8,10,10-hexamethyl-5-dodecylamino]benzoyl, 4-(2',3'-dihydrophytyloxy)-2-methylbenzoyl, 4-(2',3'-dihydrophytyloxy)benzoyl, 4-di(2',3'-dihydrophytyloxy)benzoyl, 3,5-di(2',3'-dihydrophytyloxy)benzoyl, 2-[3',4',5'-tri(2",3"-dihydrophytyloxy)benzyloxy]-4-methoxybenzoyl, 3,4,5-tri(2',3'-dihydrophytyloxy)benzoyl, 3,4,5-tri[3,4,5-tri(2',3'-dihydrophytyloxy)benzyloxy]benzoyl, or 2,4-di(2-decyltetradecyloxy)benzoyl.

[0477] Specific examples 6 of the pseudo solid phase protecting group (III) having a branched aliphatic hydrocarbon group having 10 or more carbon atoms (which is used to protect a carboxyl group) include groups wherein LY is a single bond, and Z is 4-(2',3'-dihydrophytyloxy)benzyl, 4-(3,7,11-trimethyldodecyloxy)benzyl, 4-[2,2,4,8,10,10-hexamethyl-5-dodecylamino]benzyl, 4-(2',3'-dihydrophytyloxy)-2-methylbenzyl, 4-(2',3'-dihydrophytyloxy)-2-methylbenzyl, 2-[3',4',5'-tri(2",3'-dihydrophytyloxy)benzyl]-4-methoxybenzyl, 3,4,5-tri(2',3'-dihydrophytyloxy)benzyl, 3,4,5-tri[3,4,5-tri(2',3'-dihydrophytyloxy)benzyl]benzyl, or 2,4-di(2-decyltetradecyloxy)benzyl.

[0478] The pseudo solid phase protecting group (III) is preferably any one of Specific Examples 1 to Specific Examples 6 of the above-mentioned pseudo solid phase protecting group (III), and more preferably any one of Specific Examples 1 to Specific Examples 3.

[0479] The pseudo solid phase protecting group (III) is still more preferably

[0480] Such a group, wherein L is a succinyl group or a group represented by formula (a1') (in formula (a1'), R 8 and R 9 Each independently is C 1-10alkyl, L1 is a divalent phenylene group, L2 is a single bond), and YZ is 3,4,5-tri(octadecyloxy)benzyloxy, 2,4-di(octadecyloxy)benzyloxy, 2,4-di(docosyloxy)benzyloxy, 3,5-di(docosyloxy)benzyloxy, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy, 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy, 3,4,5-tri(octadecyloxy)benzylamino, 2,4-di(docosyloxy)benzylamino, 3,5-di(docosyloxy)benzylamino, di(4-docosyloxy)benzylamino phenyl)methylamino, 4-methoxy-2-[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino, 4-methoxy-2-[3',4',5'-tri(octadecyloxy)cyclohexylmethoxy]benzylamino, 2,4-di(dodecyloxy)benzylamino, phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino, bis[4-(12-docosyloxydodecyloxy)phenyl]methylamino, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino, or 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino;

[0481] Such a group, wherein LY is succinyl-1,4-piperazinediyl, and Z is 3,4,5-tri(octadecyloxy)benzoyl, 2,4-di(octadecyloxy)benzoyl, 2,4-di(docosyloxy)benzoyl, 3,5-di(docosyloxy)benzoyl, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzoyl, or 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzoyl; or

[0482] Such a group, wherein LY is a single bond, and Z is 3,4,5-tri(octadecyloxy)benzoyl, 2,4-di(octadecyloxy)benzoyl, 2,4-di(docosyloxy)benzoyl, 3,5-di(docosyloxy)benzoyl, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzoyl, or 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzoyl.

[0483] The pseudo solid phase protecting group (III) is further preferably

[0484] Such a group, wherein L is a succinyl group or a group represented by formula (a1') (in formula (a1'), R 8 and R 9 Each independently is C 1-10alkyl, L1 is a divalent phenylene group, L2 is a single bond), and YZ is 3,4,5-tri(octadecyloxy)benzyloxy, 3,5-di(docosyloxy)benzyloxy, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy, 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy, 3,4,5-tri(octadecyloxy)benzylamino, 2,4-di(docosyloxy)benzylamino, 3,5-di(docosyloxy)benzylamino, di(4-docosyloxyphenyl)methylamino, 4-methoxy-2- -[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino, 4-methoxy-2-[3',4',5'-tri(octadecyloxy)cyclohexylmethoxy]benzylamino, 2,4-di(dodecyloxy)benzylamino, phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino, bis[4-(12-docosyloxydodecyloxy)phenyl]methylamino, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino, or 3,4,5-tris[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino; or

[0485] A group wherein LY is a single bond and Z is 3,4,5-tri(octadecyloxy)benzoyl.

[0486] The pseudo solid phase protecting group (III) is particularly preferably

[0487] Such a group, wherein L is succinyl, and YZ is 3,4,5-tri(octadecyloxy)benzyloxy, 3,5-di(docosyloxy)benzyloxy, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy, 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy, 3,4,5-tri(octadecyloxy)benzylamino, 2,4-di(docosyloxy)benzylamino, 3,5-di(docosyloxy)benzylamino, di(4-docosyloxyphenyl)methylamino, 4-methoxy-2-[ 3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino, 4-methoxy-2-[3',4',5'-tri(octadecyloxy)cyclohexylmethoxy]benzylamino, 2,4-di(dodecyloxy)benzylamino, phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino, bis[4-(12-docosyloxydodecyloxy)phenyl]methylamino, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino, or 3,4,5-tris[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino; or

[0488] A group wherein LY is a single bond and Z is 3,4,5-tri(octadecyloxy)benzoyl.

[0489] The pseudo solid phase protecting group (III) is most preferably a group wherein L is succinyl, and YZ is 3,4,5-tri(octadecyloxy)benzyloxy, 3,5-di(docosyloxy)benzyloxy, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy, 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy, 3,4,5-tri(octadecyloxy)benzylamino, 2,4-di(docosyloxy)benzylamino, 3,5-di(docosyloxy)benzylamino, di(4-docosyloxyphenyl)methylamino, 4 -methoxy-2-[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino, 4-methoxy-2-[3',4',5'-tri(octadecyloxy)cyclohexylmethoxy]benzylamino, 2,4-di(dodecyloxy)benzylamino, phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino, bis[4-(12-docosyloxydodecyloxy)phenyl]methylamino, 3,5-bis[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino, or 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino.

[0490] The compound used to form a pseudo solid phase protecting group can be prepared by a method known per se (e.g., the method described in the above-mentioned Patent Document 1 or 2, esterification, silylation, etc.) or a method analogous thereto. For example, an alcohol compound or an amine compound represented by the formula: ZYH used to form a pseudo solid phase protecting group can be prepared by the following steps or steps analogous thereto.

[0491]

[0492] wherein Q' is -O-, -S-, -C(=O)O- or -NH-, R g is a hydrogen atom, OR h Group (where R h Alkyl such as C 1-6 alkyl, etc., aralkyl such as benzyl, etc., and similar groups) or a group represented by formula (a3):

[0493]

[0494] The symbols are as defined above,

[0495] Y1 is a leaving group such as a halogen atom, etc., and the other symbols are as defined above.

[0496] Step (a)

[0497] In this step, R 5 Introduce Q'H (wherein Q' is -O-, -S-, -C(=O)O- or -NH-) of the compound represented by formula (X) to prepare the compound represented by formula (Xa). When Q' is -O-, -S- or -NH-, the corresponding R 5 halide (chloride, bromide or iodide), corresponding to R 5 The carboxylic acid or acyl halide or the corresponding R 5 The reaction is carried out in the presence or absence of a base in a solvent that does not affect the reaction. In addition, when Q' is -O-, the reaction can be carried out under Mitsunobu reaction conditions, wherein compound (X) is reacted with a sulfonyloxylated product corresponding to R 5 In the presence of triphenylphosphine and diisopropyl azodicarboxylate, the hydroxide is reacted. In addition, when Q' is -C(=O)O-, for example, compound (Xa) can be prepared by reacting compound (X) with a corresponding 5 The amine or hydroxide is reacted in the presence of a condensing agent to synthesize it.

[0498] Examples of the base include alkali metal salts such as sodium carbonate, sodium hydrogen carbonate, potassium carbonate, potassium tert-butoxide and the like; alkali metal hydrides such as sodium hydride, potassium hydride and the like; amines such as pyridine, triethylamine, N,N-dimethylaniline, 1,8-diazabicyclo[5.4.0]-7-undecene and the like, among which sodium carbonate, potassium carbonate, sodium hydride and the like are preferred.

[0499] Examples of solvents include aromatic hydrocarbons such as toluene, xylene, etc.; ethers such as tetrahydrofuran, dioxane, etc.; amides such as dimethylformamide, dimethylacetamide, etc.; halogenated hydrocarbons such as chloroform, dichloromethane, etc.; nitriles such as acetonitrile, etc., N-methylpyrrolidone, etc. or mixtures thereof, among which dimethylformamide, tetrahydrofuran, toluene, N-methylpyrrolidone, etc. are preferred.

[0500] The reaction temperature is preferably 50-150° C., more preferably 60-130° C. The reaction time is preferably 2-30 hr, more preferably 3-10 hr.

[0501] Step (b)

[0502] In this step, compound (Xa) is reduced to obtain a compound represented by formula (XI-a). This reduction reaction can be carried out by a method using a reducing agent.

[0503] Examples of the reducing agent to be used for this reduction reaction include metal hydrides (sodium borohydride, lithium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, dibutylaluminum hydride, aluminum hydride, lithium aluminum hydride, etc.) and the like, among which sodium borohydride, dibutylaluminum hydride, and the like are preferred.

[0504] This reaction is carried out in a solvent that does not affect the reaction. Examples of solvents include alcohols such as methanol, ethanol, etc.; ethers such as diethyl ether, tetrahydrofuran, dioxane, etc.; aromatic hydrocarbons such as toluene, xylene, etc.; and mixtures thereof, among which tetrahydrofuran, toluene, etc. are preferred. The reaction temperature is preferably 0-100°C, more preferably 30-70°C. The reaction time is preferably 1-24 hr, more preferably 2-5 hr.

[0505] Step (c)

[0506] In this step, compound (Xa) (wherein R g Not a hydrogen atom or OR h Group) reduction.

[0507] Step (d-1)

[0508] In this step, compound (Xa) (wherein R g is a hydrogen atom) to prepare a compound represented by formula (XI'-a).

[0509] The oximation reaction is carried out by reacting the compound (Xa) with an acid addition salt of hydroxylamine in the presence of a base in a solvent that does not influence the reaction.

[0510] Examples of the acid addition salt of hydroxylamine include inorganic acid salts such as hydrochloride, sulfate, nitrate and the like; organic acid salts such as acetate, trifluoroacetate, methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate and the like, and particularly preferably hydrochloride.

[0511] Examples of the base include alkali metal salts such as potassium hydroxide, sodium hydroxide, sodium bicarbonate, potassium carbonate, etc.; organic amines such as pyridine, triethylamine, diisopropylethylamine, N,N-dimethylaniline, 1,8-diazabicyclo[5.4.0]undec-7-ene, etc. and the like, among which triethylamine, diisopropylethylamine, etc. are preferred.

[0512] Examples of the solvent include halogenated hydrocarbons such as chloroform, dichloromethane and the like; aromatic hydrocarbons such as toluene, xylene and the like; ethers such as tetrahydrofuran, dioxane and the like; and / or mixtures thereof, among which dichloromethane, chloroform, toluene and the like are preferred.

[0513] The reaction temperature is preferably 10-100° C., more preferably 20-60° C. The reaction time is preferably 0.5-30 hr, more preferably 2-20 hr.

[0514] Step (d-2)

[0515] In this step, compound (XI'-a) is reduced by catalytic hydrogenation reaction in the presence of a metal catalyst such as palladium-carbon, Raney nickel, or using a reducing agent such as metal hydride similar to those in the above step (b) to prepare a compound represented by formula (XI-b).

[0516] Compound (XI-b) can also be produced through step (d-3), step (d-4) and step (d-5).

[0517] Step (d-3)

[0518] In this step, compound (XI-a) is halogenated with, for example, a chlorinating agent such as acetyl chloride, thionyl chloride, etc. or a brominating agent such as acetyl bromide, phosphorus tribromide, diphenylphosphine / bromine, etc. to prepare a compound represented by formula (XI'-b).

[0519] Examples of the solvent include halogenated hydrocarbons such as chloroform, dichloromethane, etc.; aromatic hydrocarbons such as toluene, xylene, etc.; ethers such as tetrahydrofuran, dioxane, etc.; and mixtures thereof, among which preferred are chloroform, tetrahydrofuran, toluene, etc. The reaction temperature is preferably 10-150° C., more preferably 30-80° C. The reaction time is preferably 0.5-30 hr, more preferably 2-20 hr.

[0520] Step (d-4)

[0521] In this step, compound (XI'-b) is azidated with an azidating agent such as sodium azide to prepare a compound represented by formula (XI'-c). This reaction is carried out by reacting compound (XI'-b) with the azidating agent in a solvent that does not affect the reaction.

[0522] Examples of solvents include halogenated hydrocarbons such as chloroform, dichloromethane, etc.; aromatic hydrocarbons such as toluene, xylene, etc.; ethers such as tetrahydrofuran, dioxane, etc.; amides such as N,N-dimethylformamide, etc.; and mixtures thereof, among which chloroform, N,N-dimethylformamide, etc. are preferred. The reaction temperature is preferably 10-150°C, more preferably 20-100°C. The reaction time is preferably 0.5-30 hr, more preferably 2-20 hr.

[0523] Step (d-5)

[0524] In this step, compound (XI'-c) is aminated to prepare compound (XI-b). This reaction is carried out by reacting compound (XI'-c) with triphenylphosphine in the presence of water in a solvent that does not affect the reaction or by catalytic hydrogenation reduction.

[0525] The amount of triphenylphosphine to be used is preferably 1-10 mol, particularly preferably 1-5 mol, per 1 mol of compound (XI'-c). The amount of water to be used is preferably 1-10 mol, particularly preferably 1-5 mol, per 1 mol of compound (XI'-c).

[0526] Examples of the solvent include aromatic hydrocarbons such as toluene, xylene, etc.; ethers such as tetrahydrofuran, dioxane, etc.; and mixtures thereof, among which preferred are toluene, tetrahydrofuran, etc. The reaction temperature is preferably 10-150° C., more preferably 20-100° C. The reaction time is preferably 0.5-30 hr, more preferably 2-20 hr.

[0527] Step (d-6)

[0528] In this step, compound (XI'-b) is reacted with RNH2 (R is as defined above) to prepare a compound represented by formula (XI-c) (wherein Y is a -NHR group). In this step, compound (XI'-b) is reacted with an amine represented by R-NH2 in the presence of a base such as a tertiary amine (triethylamine, diisopropylethylamine, etc.) (if necessary) in a solvent that does not affect the reaction.

[0529] Examples of solvents include aromatic hydrocarbons such as toluene, xylene, etc.; ethers such as tetrahydrofuran, dioxane, etc.; and halogenated hydrocarbons such as chloroform, dichloromethane, etc. and mixtures thereof, among which preferred are toluene, tetrahydrofuran, chloroform, etc. The reaction temperature is preferably 10-100° C., more preferably 20-60° C. The reaction time is preferably 0.5-30 hr, more preferably 2-20 hr.

[0530] Step (d-7)

[0531] In this step, compound (XI-d) is reacted with a compound having a -CONH2 group or a -OCONH2 group, and then treated with a base to prepare compound (XI-e). The reaction of compound (XI-d) with a compound having a -CONH2 group or a -OCONH2 group is carried out in a solvent that does not affect the reaction under an acid catalyst.

[0532] Examples of the acid catalyst include methanesulfonic acid, trifluoromethanesulfonic acid, toluenesulfonic acid, etc., among which methanesulfonic acid and toluenesulfonic acid are preferred. The amount of the acid catalyst to be used is preferably 0.05-0.5 mol, particularly preferably 0.1-0.3 mol, per 1 mol of compound (XI-d).

[0533] Examples of compounds having a -CONH2 group or a -OCONH2 group include Fmoc-NH2, HCONH2, CF3CONH2, AcNH2, EtOCONH2, Cbz-NH2, etc., among which preferred are Fmoc-NH2, EtOCONH2, etc. As used herein, "Fmoc-" refers to a 9-fluorenylmethoxycarbonyl group (hereinafter also referred to as an Fmoc group), and "Cbz-" refers to a benzyloxycarbonyl group (hereinafter also referred to as a Cbz group).

[0534] As a reagent to be used as a starting material for step (a) [i.e., corresponding to R 5 The hydroxide, halide, alkylsulfonyloxylation product (e.g., methanesulfonyloxylation product, etc.) or arylsulfonyloxylation product (e.g., p-toluenesulfonyloxylation product, etc.), hereinafter referred to as "reagent of step (a)"], commercially available products can be used. In addition, the reagent of step (a) can be prepared, for example, by the following method:

[0535] (1) Corresponding to R 5 Halogenation, alkylsulfonyloxylation or arylsulfonyloxylation of the hydroxide of

[0536] (2) Corresponding to R 5 Reduction reaction of the unsaturated hydroxide of the present invention (for example, catalytic hydrogenation reaction in the presence of a metal catalyst such as platinum-carbon (Pt / C), palladium-carbon (Pd / C), rhodium-carbon (Rh / C), Raney nickel, etc.), followed by halogenation, alkylsulfonyloxylation or arylsulfonyloxylation.

[0537] In the preparation of the reagent of step (a), examples of the reagent for converting the hydroxyl group into a leaving group include halogenating agents such as chlorinating agents (thionyl chloride, N-chlorosuccinimide (NCS), etc.), brominating agents (hydrobromic acid, acetyl bromide, N-bromosuccinimide (NBS), phosphorus tribromide, diphenylphosphine / bromine, etc.), etc., in addition to these, alkylsulfonylating agents (such as methanesulfonyl chloride, trifluoromethanesulfonyl chloride, etc.), arylsulfonylating agents (such as benzenesulfonyl chloride, p-toluenesulfonyl chloride, etc.) and the like, among which preferred are halogenating agents such as thionyl chloride, hydrobromic acid, etc.

[0538] This reaction is carried out in a solvent that does not affect the reaction, and examples of the solvent include water; halogenated hydrocarbons such as chloroform, dichloromethane, etc.; aromatic hydrocarbons such as benzene, toluene, xylene, etc.; nitriles such as acetonitrile, propionitrile, etc.; ethers such as tetrahydrofuran, 1,4-dioxane, diethyl ether, etc., among which water, halogenated hydrocarbons such as chloroform, etc. are preferred. The reaction temperature is preferably 10-120°C, more preferably 50-100°C. The reaction time is preferably 1-72 hr, more preferably 3-24 hr.

[0539] The compound represented by the formula: ZYH (wherein Q' is a single bond) can also be prepared by, for example, the following method. 5 The introduction can be done in the following ways:

[0540] (1) Use the method corresponding to R 5 halide (chloride, bromide or iodide), or the compound corresponding to R 5 Friedel-Crafts reaction of carboxylic acids or acyl halides,

[0541] (2) a method comprising subjecting a compound corresponding to the above-mentioned compound (X) (a compound in which Q'H is replaced by a -CHO group) to carbon homologation by Wittig reaction, followed by catalytic hydrogenation, or the like; or

[0542] (3) Conventionally used organic synthesis reactions such as cross coupling using a metal catalyst.

[0543] In each of the above schemes, reaction reagents and the like are shown for the sake of convenience and may be appropriately changed within the range defined above.

[0544] [Organic compound protected by pseudo solid phase protecting group of the present invention]

[0545] The organic compound protected by an organic group having one or more aliphatic hydrocarbon groups having 10 or more carbon atoms (the pseudo-solid-phase protecting group of the present invention) is not particularly limited as long as it can be precipitated from a solvent, and organic compounds having functional groups such as hydroxyl (-OH), thiol (-SH), carboxyl (-COOH), amino (-NHR), imino (-NH-), carboxamide (-CONHR") (wherein R" is a hydrogen atom or a hydrocarbon group), etc. (wherein the functional group is protected by the pseudo-solid-phase protecting group of the present invention) (hereinafter sometimes referred to as "protected organic compound") can be used. The protected organic compound may be further protected by a protecting group different from the pseudo-solid-phase protecting group (e.g., a protecting group for nucleic acid synthesis). Examples of the protected organic compound include nucleosides, nucleotides, amino acids, peptides and the like disclosed in WO 2012 / 157723, WO 2012 / 157723, WO 2014 / 189142, JP-A-2010-116418, WO 2010 / 104169, WO 2010 / 113939, WO 2011 / 078295, WO 2012 / 029794, JP-A-2009-185063, JP-A-2010-275254 and the like.

[0546] Preferred protected organic compound is the nucleoside, nucleotide or oligonucleotide that are optionally further used to the protecting group protection of nucleic acid synthesis, or the amino acid or peptide that are optionally further used to the protecting group protection of peptide synthesis.Preferably, in the above-mentioned nucleoside, nucleotide or oligonucleotide, at least one group selected from the amino and imino groups of nucleic acid bases, 2' and 3' hydroxyls of ribose residues and 3' hydroxyls of deoxyribose residues is protected by pseudo solid phase protecting group of the present invention.When the above-mentioned nucleoside, nucleotide or oligonucleotide is morpholino nucleoside, morpholino nucleotide or morpholino oligonucleotide, the 5' hydroxyl of morpholine residue instead of the above-mentioned hydroxyl can be protected by pseudo solid phase protecting group.

[0547] More preferably, the protected organic compound is a nucleoside or an oligonucleotide, wherein at least one group selected from the amino and imino groups of nucleic acid bases, the 2' and 3' hydroxyl groups of ribose residues, the 3' hydroxyl group of deoxyribose residues, and the 5' hydroxyl group of morpholine residues is protected by the pseudo-solid phase protecting group of the present invention, and other groups are optionally further protected by protecting groups used for nucleic acid synthesis.

[0548] Further preferred protected organic compounds are nucleosides or oligonucleotides, in which at least one group selected from the amino and imino groups of nucleic acid bases, the 2' and 3' hydroxyl groups of ribose residues, and the 3' hydroxyl group of deoxyribose residues is protected by the pseudo-solid phase protecting group of the present invention, and other groups are optionally further protected by protecting groups used for nucleic acid synthesis.

[0549] Particularly preferred protected organic compounds are nucleosides or oligonucleotides, in which at least one group selected from the amino and imino groups of nucleic acid bases, the 3'-hydroxyl group of ribose residues, and the 3'-hydroxyl group of deoxyribose residues is protected by the pseudo-solid phase protecting group of the present invention, and other groups are optionally further protected by protecting groups used for nucleic acid synthesis.

[0550] The most preferred protected organic compound is a nucleoside or oligonucleotide, wherein the 3' hydroxyl group of a ribose residue or the 3' hydroxyl group of a deoxyribose residue is protected by the pseudo solid phase protecting group of the present invention, and other groups are optionally further protected by protecting groups used for nucleic acid synthesis.

[0551] [precipitation]

[0552] The present invention also provides a method for precipitating a protected organic compound from a solvent by using the precipitation accelerator of the present invention (hereinafter sometimes referred to as "the precipitation method of the present invention") and a precipitation mixture obtained by this method, the precipitation mixture comprising the precipitation accelerator and the protected organic compound. The above-mentioned solvent is preferably a solvent comprising a polar solvent, more preferably a mixed solvent of a polar solvent and a non-polar solvent.

[0553] Due to the pseudo solid phase protecting group (which is a hydrophobic group), the organic compound protected by the pseudo solid phase protecting group of the present invention shows improved solubility in non-polar solvents and reduced solubility in polar solvents. The organic compound protected by this pseudo solid phase protecting group is precipitated, for example, by adding a polar solvent to a solution in which the organic compound is dissolved in a non-polar solvent to achieve solid-liquid separation. The precipitation promoter of the present invention can promote the precipitation of the organic compound protected by the pseudo solid phase protecting group from the solvent and can improve its recovery rate. In addition, the deprotected organic compound can also be obtained by removing the pseudo solid phase protecting group after obtaining the target product (i.e., the organic compound protected by the pseudo solid phase protecting group).

[0554] Examples of the polar solvent include alcohol solvents such as methanol, ethanol, isopropanol, etc.; nitrile solvents such as acetonitrile, propionitrile, etc.; ketone solvents such as acetone, 2-butanone, etc.; polar ether solvents such as 1,4-dioxane, tetrahydrofuran, etc.; amide solvents such as dimethylformamide, dimethylacetamide, N-methylpiperidone, etc.; sulfoxide solvents such as dimethyl sulfoxide, etc.; water, etc. The polar solvent can use only one or two or more thereof can be used in combination. As a polar solvent, acetonitrile is particularly preferred. In the synthesis of oligonucleotides, from the perspective of precipitation efficiency, there is a tendency for the recovery rate to increase when methanol is used as a polar solvent. Since methanol inhibits the coupling reaction in the phosphoramidite method, methanol needs to be completely removed from the precipitated oligonucleotide. However, drying takes a long time and it is problematic that it is difficult to judge whether methanol remains in the system. On the other hand, acetonitrile tends to reduce the precipitation efficiency, but does not inhibit the coupling reaction in the phosphoramidite method. Therefore, the oligonucleotide precipitated using acetonitrile does not require strict drying and judgment of residual solvents. Using the precipitation promoter of the present invention, the target product can be precipitated and recovered in high yield even when acetonitrile is used.

[0555] Examples of the non-polar solvent include halogen solvents such as chloroform, dichloromethane, 1,2-dichloroethane, etc.; aromatic solvents such as benzene, toluene, xylene, mesitylene, etc.; ester solvents such as ethyl acetate, isopropyl acetate, etc.; aliphatic solvents such as hexane, pentane, heptane, octane, nonane, cyclohexane, etc.; non-polar ether solvents such as diethyl ether, cyclopentyl methyl ether, tert-butyl methyl ether, etc. The non-polar solvent can be used alone or in combination of two or more thereof. As the non-polar solvent, halogen solvents, aromatic solvents, ester solvents, aliphatic solvents and combinations thereof are preferred; dichloromethane, chloroform, 1,2-dichloroethane, benzene, toluene, xylene, mesitylene, hexane, pentane, heptane, nonane, cyclohexane, ethyl acetate, isopropyl acetate and combinations thereof are more preferred; chloroform, dichloromethane, toluene and combinations thereof are more preferred.

[0556] In order to fully precipitate the protected organic compound, the amount of the precipitation promoter of the present invention to be used is preferably not less than 0.1 molar equivalent, more preferably not less than 0.5 molar equivalent, and further preferably not less than 1 molar equivalent relative to the protected organic compound. Although the upper limit is not particularly limited, from the perspective of cost, the amount of the precipitation promoter of the present invention to be used is preferably not more than 10 molar equivalents, more preferably not more than 5 molar equivalents, and further preferably not more than 2.5 molar equivalents relative to the protected organic compound.

[0557] The amount of the polar solvent in the solvent containing the polar solvent (preferably acetonitrile) is preferably not less than 0.5 mL, more preferably not less than 1 mL, further preferably not less than 2 mL, preferably not more than 20 mL, more preferably not more than 10 mL, and further preferably not more than 8 mL relative to each 1 mL of the solvent other than the polar solvent, so as to improve the recovery rate of the protected organic compound. The amount of the polar solvent is the preferred range when the protected organic compound is precipitated. For example, after concentrating the solution containing the protected organic compound and the solvent other than the polar solvent, the polar solvent is added within the above range to precipitate the organic compound. In addition, a solution containing the protected organic compound, the solvent other than the polar solvent, and the polar solvent is prepared so that the amount of the polar solvent is lower than the above lower limit, and it is concentrated to adjust the amount of the polar solvent to not less than the above lower limit, so that the organic compound can be precipitated.

[0558] The temperature during precipitation of the protected organic compound is preferably not lower than -10°C, more preferably not lower than 0°C, further preferably not lower than 5°C, preferably not higher than 40°C, more preferably not higher than 30°C, further preferably not higher than 25°C.

[0559] After adjusting to the above-mentioned preferred temperature, the standing time (i.e., the time for precipitation) of the suspension of the protected organic compound is preferably not less than 5 min, more preferably not less than 15 min, further preferably not less than 30 min, preferably not more than 72 hr, more preferably not more than 16 hr, and further preferably not more than 3 hr.

[0560] [Method for preparing oligonucleotide]

[0561] The precipitation method of the present invention is particularly useful for the preparation method of oligonucleotide. Therefore, the present invention also provides the preparation method of oligonucleotide comprising the precipitation method of the present invention. The example of this preparation method includes the method comprising the following steps: adding a polar solvent (preferably acetonitrile) in a reaction solution comprising an oligonucleotide and a precipitation promoter of the present invention, and separating the step of a precipitation mixture comprising an oligonucleotide and a precipitation promoter from the reaction solution, wherein at least one group in the oligonucleotide is protected by a pseudo solid phase protecting group of the present invention and other groups are optionally further protected by a protecting group used in nucleic acid synthesis. The preparation method of oligonucleotide of the present invention is preferably carried out by a phosphoramidite method.

[0562] A preferred embodiment of the method for producing an oligonucleotide of the present invention is a method for producing an oligonucleotide, comprising performing a production cycle comprising the following steps (1) to (3) once or repeatedly a plurality of times by a phosphoramidite method, wherein the following step (4) is included in the first cycle, the following step (5) is included in each cycle, and the following step (6) is included in each cycle except the final cycle,

[0563] (1) adding an acid to a reaction solution containing a nucleoside or oligonucleotide (hereinafter sometimes referred to as "nucleoside or oligonucleotide containing a temporary protecting group") in a non-polar solvent to deprotect the temporary protecting group of the 5'-hydroxyl group, and neutralizing with a base to obtain a reaction solution containing a free form of the 5'-hydroxyl group (deprotection step),

[0564] In the nucleoside or oligonucleotide, at least one group selected from the group consisting of the amino and imino groups of the nucleic acid bases, the 2' and 3' hydroxyl groups of the ribose residues, and the 3' hydroxyl group of the deoxyribose residues is protected by the pseudo solid phase protecting group of the present invention, the 5' hydroxyl group is protected by a temporary protecting group removable under acidic conditions, and the other groups are optionally further protected by protecting groups used in nucleic acid synthesis;

[0565] (2) a step of adding a nucleoside or oligonucleotide (hereinafter sometimes referred to as "phosphoramidated nucleoside or oligonucleotide") to a reaction solution containing a free 5'-hydroxyl group in a non-polar solvent to obtain a reaction solution containing a triester phosphite compound (condensation step),

[0566] In the nucleoside or oligonucleotide, the 3'-hydroxyl group is phosphoramidated, and the 5'-hydroxyl group is protected by a temporary protecting group that can be removed under acidic conditions, and other groups are optionally further protected by protecting groups used in nucleic acid synthesis;

[0567] (3) a step of adding an oxidizing agent or a sulfurizing agent to a reaction solution containing a triester phosphite compound in a nonpolar solvent to obtain a reaction solution containing an oligonucleotide (hereinafter sometimes referred to as "product oligonucleotide") (oxidation or sulfurization step),

[0568] In the oligonucleotide, at least one group selected from the group consisting of the amino and imino groups of the nucleic acid bases, the 2' and 3' hydroxyl groups of the ribose residues, and the 3' hydroxyl group of the deoxyribose residues is protected by the pseudo solid phase protecting group of the present invention, and the 5' hydroxyl group is protected by a temporary protecting group removable under acidic conditions, and the other groups are optionally further protected by protecting groups used in nucleic acid synthesis;

[0569] (4) a step of adding the precipitation accelerator of the present invention to the reaction solution at any time before step (1), between steps (1) and (2), between steps (2) and (3), and after step (3) (precipitation accelerator addition step);

[0570] (5) after step (4), and at any time between steps (1) and (2), between steps (2) and (3), and after step (3), a step of adding a polar solvent (preferably acetonitrile) to the reaction solution containing the precipitation accelerator to separate a precipitation mixture containing the 5'-hydroxyl free body, the triester phosphite compound or the oligonucleotide, and the precipitation accelerator from the reaction solution (precipitation and solid-liquid separation step);

[0571] (6) A step of adding a non-polar solvent to the precipitate mixture obtained in step (5) to prepare a reaction solution (dissolving step).

[0572] The following explains each step in the above order.

[0573] Step (1) (deprotection step)

[0574] In this step, the temporary protecting group is removed from the nucleoside or oligonucleotide containing the temporary protecting group by adding acid to obtain the 5' hydroxyl free body. Here, the temporary protecting group refers to a protecting group that can be removed under acidic conditions where the pseudo solid phase protecting group of the present invention is not removed.

[0575] This step is carried out in a nonpolar solvent. As the nonpolar solvent, the above-mentioned nonpolar solvents can be mentioned. The same applies to the nonpolar solvents to be used in the steps after this step.

[0576] Although the concentration of the nucleoside or oligonucleotide having a temporary protecting group in the solution in this step is not particularly limited as long as they are dissolved in the solvent, it is preferably 1 to 30 wt %.

[0577] Although there is no particular restriction on the acid to be used in this step, as long as good deprotection can be achieved, trifluoroacetic acid, dichloroacetic acid, trifluoromethanesulfonic acid, trichloroacetic acid, methanesulfonic acid, hydrochloric acid, acetic acid, p-toluenesulfonic acid etc. are preferably used. Because good reaction can be achieved, trifluoroacetic acid, dichloroacetic acid, trifluoromethanesulfonic acid, trichloroacetic acid are more preferably used, and then trifluoroacetic acid, dichloroacetic acid, trifluoromethanesulfonic acid are preferred, and trifluoroacetic acid, trifluoromethanesulfonic acid are also more preferably used, and trifluoroacetic acid is particularly preferred. These acids can be diluted with the above-mentioned non-polar solvent. When using the above-mentioned acid, it can be combined with a specific base before use to appropriately adjust the acidity. The amount of the acid to be used in this step is, for example, 1 to 100 mol, preferably 1 to 40 mol, relative to every 1 mol of nucleosides or oligonucleotides containing temporary protecting groups.

[0578] Although the reaction temperature in this step is not particularly limited as long as the reaction proceeds, it is preferably -10°C to 50°C, more preferably 0°C to 40°C. The reaction time varies depending on the nucleoside or oligonucleotide containing a temporary protecting group to be used, the kind of acid, the kind of nonpolar solvent, the reaction temperature and the like, but it is, for example, 5 min to 5 hr.

[0579] In order to continuously carry out this step, the subsequent condensation step and the oxidation or sulfurization step in a solution, it is preferred to use a cation scavenger in this step during or after the removal reaction of the temporary protecting group of the 5'-hydroxyl group of the nucleoside or oligonucleotide containing the temporary protecting group.

[0580] Although there are no particular limitations on the cation scavenger, as long as it is not re-protected by the removed temporary protecting group (returns to the starting material) and does not undergo side reactions with the deprotected functional group, pyrrole derivatives such as pyrrole, 2-methylpyrrole, 3-methylpyrrole, 2,3-dimethylpyrrole, 2,4-dimethylpyrrole, etc., and indole derivatives such as indole, 4-methylindole, 5-methylindole, 6-methylindole, 7-methylindole, 5,6-dimethylindole, 6,7-dimethylindole, etc. can be used. Since a good cation capturing effect can be obtained, pyrrole, 3-methylpyrrole, 2,4-dimethylpyrrole, indole, 4-methylindole, 5-methylindole, 6-methylindole, 7-methylindole, 5,6-dimethylindole, 6,7-dimethylindole, etc. are preferred, pyrrole, 3-methylpyrrole, indole are more preferred, pyrrole and indole are further preferred, and pyrrole is particularly preferred.

[0581] The amount of the cation scavenger to be used in this step is, for example, 1 to 50 mol, preferably 5 to 20 mol, per 1 mol of the nucleoside or oligonucleotide having a temporary protecting group.

[0582] When an acid to be used as a deprotecting agent is present in the condensation step as the next step, it causes deprotection of the temporary protecting group of the phosphoramidite nucleoside or oligonucleotide used in step (2). Therefore, the acid needs to be neutralized and removed.

[0583] In this step, an organic base is preferably used for neutralization of the acid. As the organic base, pyridine, 2,4,6-trimethylpyridine, benzimidazole, 1,2,4-triazole, N-phenylimidazole, 2-amino-4,6-dimethylpyrimidine, 1,10-phenanthroline, imidazole, N-methylimidazole, 2-chlorobenzimidazole, 2-bromobenzimidazole, 2-methylimidazole, 2-phenylbenzimidazole, N-phenylbenzimidazole, 5-nitrobenzimidazole are preferred, and pyridine, 2,4,6-trimethylpyridine, benzoimidazole, 1,2,4-triazole, N-phenylimidazole, 2-amino-4,6-dimethylpyrimidine, 1,10-phenanthroline, imidazole, N-methylimidazole, 2-chlorobenzimidazole, 2-bromobenzimidazole, 2-methylimidazole, 2-phenylbenzimidazole, N-phenylbenzimidazole, 5-nitrobenzimidazole are more preferred. Imidazole, 1,2,4-triazole, N-phenylimidazole, N-methylimidazole, 2-amino-4,6-dimethylpyrimidine, 1,10-phenanthroline, further preferably pyridine, 2,4,6-trimethylpyridine, benzimidazole, 1,2,4-triazole, N-phenylimidazole, particularly preferably pyridine, 2,4,6-trimethylpyridine, benzimidazole, 1,2,4-triazole, and most preferably pyridine, 2,4,6-trimethylpyridine, benzimidazole.

[0584] The amount of the base to be used in this step is, for example, 1 to 10 mol, preferably 1 to 3 mol, per 1 mol of the acid.

[0585] In this step, particularly preferred combinations of the acid and the organic base are trifluoroacetic acid and pyridine, trifluoroacetic acid and 2,4,6-trimethylpyridine, or trifluoromethanesulfonic acid and benzimidazole.

[0586] The temporary protecting group contained in the nucleoside or oligonucleotide containing a temporary protecting group to be used in this step is not particularly limited as long as it can be deprotected under acidic conditions and can be used as a hydroxyl protecting group. For example, trityl, 9-(9-phenyl)xanthenyl, 9-phenylthioxanthenyl, di(C 1-6 Alkoxy) triphenylmethyl such as 1,1-bis(4-methoxyphenyl)-1-phenylmethyl (dimethoxytrityl), mono(C 1-18 The temporary protecting group is preferably a monomethoxytrityl group, a dimethoxytrityl group, and more preferably a dimethoxytrityl group, in consideration of the ease of deprotection and availability.

[0587] In the nucleoside or oligonucleotide containing a temporary protecting group to be used in this step, at least one group selected from the amino and imino groups of the nucleic acid bases, the 3'-hydroxyl group of the ribose residue, and the 3'-hydroxyl group of the deoxyribose residue is preferably protected by the pseudo-solid phase protecting group of the present invention, and the 3'-hydroxyl group of the ribose residue or the 3'-hydroxyl group of the deoxyribose residue is more preferably protected by the pseudo-solid phase protecting group of the present invention.

[0588] The nucleoside or oligonucleotide containing a temporary protecting group to be used in this step is further preferably a compound represented by the following formula (VI):

[0589]

[0590] in:

[0591] m" is an integer not less than 0,

[0592] m"+1 bases are each independently an optionally protected nucleic acid base,

[0593] Q' is a temporary protecting group that can be removed under acidic conditions,

[0594] m"+1 X are each independently a hydrogen atom, a halogen atom, an optionally protected hydroxyl group, or a divalent organic group bonded to the carbon atom at position 2 and the carbon atom at position 4 of a ribose ring or a deoxyribose ring (preferably a hydrogen atom, a halogen atom or an optionally protected hydroxyl group),

[0595] m” R 10 are each independently an oxygen atom or a sulfur atom,

[0596] m" WGs are each independently an electron withdrawing group, and

[0597] LYZ is as defined above.

[0598] When m" is 0, compound (VI) is a nucleoside containing a temporary protecting group; and when m" is 1 or more, compound (VI) is an oligonucleotide containing a temporary protecting group. In formula (VI), m" is preferably not more than 49, more preferably not more than 29, further preferably not more than 19, particularly preferably not more than 4, and most preferably not more than 2.

[0599] As the halogen atom in the formula (VI), a fluorine atom and a chloride ion are preferred, and a fluorine atom is more preferred.

[0600] Although the protecting group of the “optionally protected hydroxyl group” in formula (VI) is not particularly limited, for example, any protecting group described in PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd edition, JOHN WILLY & SONS (1999) etc. can be mentioned. Specifically, methyl, benzyl, p-methoxybenzyl, tert-butyl, methoxymethyl, methoxyethyl, 2-tetrahydropyranyl, ethoxyethyl, cyanoethyl, cyanoethoxymethyl, phenylcarbamoyl, 1,1-dioxothiomorpholine-4-thiocarbamoyl, acetyl, pivaloyl, benzoyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, [(triisopropylsilyl)oxy]methyl (Tom), 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) and the like can be mentioned. Among them, a triethylsilyl group, a triisopropylsilyl group, or a tert-butyldimethylsilyl group is preferred. In terms of economic efficiency and easy availability, a tert-butyldimethylsilyl group is particularly preferred.

[0601] In formula (VI), the "divalent organic group bonded to the carbon atom at position 2 and the carbon atom at position 4 of the ribose ring or deoxyribose ring" is not particularly limited as long as it is bonded to the carbon atom at position 2 and the carbon atom at position 4 of the same ribose ring or deoxyribose ring. Examples of the divalent organic group include optionally substituted C 2-7 Alkylene, and selected from -O-, -NR 13 -(R 13 A hydrogen atom or C 1-6 alkyl), -S-, -CO-, -COO-, -OCONR 14 -(R 14 A hydrogen atom or C 1-6 alkyl) and -CONR 15 -(R 15 A hydrogen atom or C 1-6 alkyl) and an optionally substituted C 1-7 Divalent organic groups formed by alkylene, etc. 1-7 Alkylene and C 2-7 Examples of the substituent which the alkylene group may have include a methylene group (CH2=).

[0602] As the "divalent organic group bonded to the carbon atom at the 2-position and the carbon atom at the 4-position", preferably an optionally substituted C 2-7 Alkylene, -ORi- (Ri is a C bonded to the 4-carbon atom 1-6 Alkylene), -O-NR 13 -Rj-(Rj is the C bonded to the 4-carbon atom 1-6 Alkylene, and R 13As defined above), -O-Rk-O-Rl-(Rk is C 1-6 Alkylene, and R1 is a C bonded to and cross-linked with the carbon atom at position 4 1-6 alkylene), and more preferably -ORi- (Ri is as defined above), -O-NR 13 -Rj-(Rj and R 13 As defined above), -O-Rk-O-Rl- (Rk and Rl are as defined above). C 1-6 The alkylene groups are each independently preferably a methylene group or an ethylene group.

[0603] As the "divalent organic group bonded to the carbon atom at the 2-position and the carbon atom at the 4-position", -O-CH2-, -O-CH2-CH2-, -O-NR 13 -CH2-(R 13 As defined above), -O-CH2-O-CH2-, and further preferably -O-CH2-, -O-CH2-CH2-, -O-NH-CH2-, -ON(CH3)-CH2-, -O-CH2-O-CH2- (wherein the left side is bonded to the carbon atom at the 2-position and the right side is bonded to the carbon atom at the 4-position).

[0604] In formula (VI), m"+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.

[0605] m” R 10 Each is preferably an oxygen atom.

[0606] Examples of the electron withdrawing group of WG include cyano, nitro, and the like, with cyano being preferred.

[0607] Compound (VI) can be synthesized by a method known per se (e.g., the method described in the above-mentioned Patent Document 1 or 2, esterification, silylation, etc.) or a method analogous thereto. For example, compound (VI) in which m" is 0 and L is a succinyl group can be synthesized by, for example, reacting a nucleoside (a) in which the 5'-hydroxyl group is protected by a temporary protecting group with succinic anhydride in the presence of a base to obtain a compound (b) in which succinic acid is introduced into the 3'-hydroxyl group, followed by dehydration condensation of the obtained compound (b) with ZYH in the presence of a condensing agent, as shown in the following formula.

[0608]

[0609] The conversion reaction from nucleoside (a) to compound (b) is advantageously carried out in a solvent inert to the reaction. Although there is no particular limitation on such solvent, as long as the reaction proceeds, halogen solvents such as dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, etc., aromatic solvents such as benzene, toluene, xylene, etc., aliphatic solvents such as pentane, hexane, heptane, octane, etc., ether solvents such as diethyl ether, tetrahydrofuran, cyclopentyl methyl ether, etc., and mixed solvents thereof are preferred. Among them, dichloromethane or chloroform is particularly preferred.

[0610] Although there is no particular limitation on the base to be used in the synthesis of compound (VI), an organic base is preferred, and triethylamine and the like are more preferred.

[0611] The dehydration condensation reaction for the synthesis of compound (VI) is advantageously carried out in a solvent that is inert to the reaction. Although there is no particular limitation on such solvents, as long as the reaction proceeds, halogenated solvents such as dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, etc., aromatic solvents such as benzene, toluene, xylene, etc., or aliphatic solvents such as pentane, hexane, heptane, octane, etc., and combinations thereof are preferred. Among them, dichloromethane and chloroform are particularly preferred.

[0612] Examples of the condensing agent used in the condensation reaction of compound (b) with ZYH include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), N-ethyl-N'-3-dimethylaminopropylcarbodiimide and its hydrochloride (EDCHCl), (benzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate (PyBop), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium-3-oxide hexafluorophosphate (HCTU), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), etc. Among them, HBTU, HCTU, N-ethyl-N'-3-dimethylaminopropylcarbodiimide and its hydrochloride (EDC HCl) are preferred.

[0613] The amount of the condensing agent to be used is, for example, 1 to 10 mol, preferably 1 to 5 mol, per 1 mol of the compound (b). The amount of ZYH to be used is, for example, 1 to 10 mol, preferably 1 to 5 mol, per 1 mol of the compound (b). Although the reaction temperature is not particularly limited as long as the reaction proceeds, it is preferably -10°C to 50°C, more preferably 0°C to 30°C. The reaction time is, for example, 30 min to 70 hr.

[0614] Compound (VI) wherein L is not succinyl can also be synthesized by performing a reaction similar to the above-mentioned synthesis method, except that the corresponding acid anhydride, the corresponding dicarboxylic acid halide, the corresponding dicarboxylic acid activated ester, etc. are used instead of succinic anhydride. In addition, compound (VI) wherein m" is 1 or more can be synthesized by repeating the 5' terminal extension process using compound (VI) wherein m" is 0 as a starting material.

[0615] Step (2) (Condensation step)

[0616] In this step, the 5'-hydroxyl free body obtained in step (1) and the phosphoramidite-modified nucleoside or oligonucleotide are condensed to obtain a phosphite triester compound.

[0617] The phosphoramidite nucleoside or oligonucleotide to be used in this step can be synthesized by a known method (MH Caruthers et al., Method in Enzymology 1987, 154, 287-313; SL Beaucage and MH Caruthers, Tetrahedron Letters 1981, 22, 1859-1862), which comprises reacting a phosphoramidite reagent. The phosphoramidite reagent is commercially available and can be easily obtained.

[0618] The phosphoramidite nucleoside or oligonucleotide to be used in this step is preferably a compound represented by the following formula (VII):

[0619]

[0620] in

[0621] q' is an integer not less than 0;

[0622] R 11 and R 12 Each independently represents an alkyl group, or a 5- or 6-membered saturated cyclic amino group formed together with an adjacent nitrogen atom. The saturated cyclic amino group optionally has an oxygen atom or a sulfur atom as a ring-constituting atom other than a nitrogen atom; and

[0623] Other symbols are as defined above.

[0624] When q' is 0, compound (VII) is a phosphoramidite nucleoside, and when q' is 1 or more, compound (VII) is a phosphoramidite oligonucleotide. In compound (VII) to be used in this step, q' is preferably not more than 49, more preferably not more than 29, further preferably not more than 19, particularly preferably not more than 4, and most preferably not more than 2.

[0625] When the condensation reaction in this step proceeds slowly, a condensation agent (eg, pyridine-trifluoroacetate, tetrazole, 5-benzylthio-1H-tetrazole, 4,5-dicyanoimidazole, etc.) may be added.

[0626] In this step, when the acidity of the reaction solution becomes high, a side reaction of removing the temporary protecting group may occur. Therefore, N-methylimidazole is preferably added to suppress the acidification of the reaction solution. The amount of N-methylimidazole to be added to adjust the acidity is preferably 0.1 to 1 mol, more preferably about 0.5 mol, per 1 mol of the organic base used for neutralization.

[0627] Similar to step (1), this step is carried out in a non-polar solvent. The explanation of the non-polar solvent is the same as in step (1).

[0628] The amount of the phosphoramidite nucleoside or oligonucleotide to be used in this step is, for example, 1 to 10 mol, preferably 1 to 5 mol, per 1 mol of the 5'-hydroxyl free form obtained in step (1).

[0629] Although the reaction temperature in this step is not particularly limited as long as the reaction proceeds, it is preferably 0° C.-100° C., and more preferably 20° C.-50° C. Although the reaction time varies depending on the kind of raw material to be used, reaction temperature and the like, it is, for example, 5 min to 24 hr.

[0630] Step (3) (Oxidation or sulfurization step)

[0631] In this step, the phosphite triester compound obtained in step (2) is reacted with an oxidizing agent or a sulfurizing agent to convert its phosphite triester bond into a phosphotriester bond or a phosphorothioate triester bond to obtain a resultant oligonucleotide.

[0632] Although the "oxidant" to be used in this step is not particularly limited, as long as it can oxidize the triester bond of phosphite to the triester bond of phosphate without oxidizing other parts, iodine, (1S)-(+)-10-camphorsulfonyloxaziridine, tert-butyl hydroperoxide (TBHP), 2-butanone peroxide, 1,1-dihydroperoxycyclododecane, bis(trimethylsilyl)peroxide, and meta-chloroperbenzoic acid are preferably used. Since a good oxidation reaction can be achieved, iodine, (1S)-(+)-10-camphorsulfonyloxaziridine, tert-butyl hydroperoxide, 2-butanone peroxide, and 1,1-dihydroperoxycyclododecane are more preferred, iodine, (1S)-(+)-10-camphorsulfonyloxaziridine, tert-butyl hydroperoxide, and 2-butanone peroxide are further preferred, iodine and tert-butyl hydroperoxide are still more preferred, and iodine is particularly preferred. The oxidant can be used after being diluted with a suitable solvent to a concentration of 0.05 to 2 M. Although there is no particular limitation on the dilution solvent as long as it is inert to the reaction, pyridine, THF, dichloromethane, water or a combination thereof can be mentioned. Among them, for example, iodine / water / pyridine-THF or iodine / pyridine-acetic acid, peroxide (TBHP) / dichloromethane or tert-butyl hydroperoxide / nonane are preferably used.

[0633] There is no particular limitation on the "vulcanizing agent" to be used in this step, as long as it can convert a phosphite triester bond into a thiophosphothioate triester bond, and preferably 3-[(N,N-dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-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), 3-amino-1,2,4-dithiazole-5-thione (ADTT), sulfur are used. Since a good reaction can be performed, 3-[(N,N-dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione (DDTT), 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent), 3H-1,2-benzodithiol-3-one, phenylacetyl disulfide (PADS) are more preferred, 3-[(N,N-dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione, 3H-1,2-benzodithiol-3-one-1,1-dioxide are further preferred, and 3-[(N,N-dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione is particularly preferred. The sulfurizing agent can be used after diluting with a suitable solvent to obtain a concentration of 0.05 to 2M. While the diluting solvent is not particularly limited as long as it is inert to the reaction, there can be mentioned, for example, dichloromethane, acetonitrile, pyridine or any mixed solvent thereof.

[0634] The amount of the oxidizing agent or the sulfiding agent to be used is, for example, 1 to 50 mol, preferably 1 to 5 mol, per 1 mol of the triester phosphite compound obtained in step (2).

[0635] Although the reaction temperature is not particularly limited as long as the reaction proceeds, it is preferably 0° C. to 100° C., and more preferably 20° C. to 50° C. Although the reaction time varies depending on the kind of the triester phosphite compound, the kind of the oxidizing agent or sulfiding agent to be used, the reaction temperature, etc., it is 1 min to 3 hr.

[0636] Step (4) (Step of adding a precipitation accelerator)

[0637] In this step, before step (5), and at any time before step (1), between steps (1) and (2), between steps (2) and (3), and after step (3), the precipitation promoter of the present invention is added to the reaction solution to promote precipitation in step (5) (precipitation and solid-liquid separation step), wherein in step (5), the precipitated oligonucleotide is separated from the reaction solution by adding a polar solvent (preferably acetonitrile) to the reaction solution between step (1) (deprotection step) and step (2) (condensation step), between step (2) and step (3) (oxidation or sulfurization step), and at any time after step (3).

[0638] In this step, only one precipitation promoter of the present invention may be added or two or more thereof may be added. The amount of the precipitation promoter of the present invention to be used in this step is preferably not less than 0.1 molar equivalent, more preferably not less than 0.5 molar equivalent, and further preferably not less than 1 molar equivalent relative to the nucleoside or oligonucleotide containing a temporary protecting group used in the initial step (1). Although there is no particular restriction on its upper limit, from the cost aspect, the amount of the precipitation promoter of the present invention to be used is preferably not more than 10 molar equivalents, more preferably not more than 5 molar equivalents, and further preferably not more than 2.5 molar equivalents relative to the nucleoside or oligonucleotide containing a temporary protecting group used in the initial step (1).

[0639] Step (5) (precipitation and solid-liquid separation step)

[0640] In this step, after step (4) (step of adding a precipitation accelerator), and at any time between step (1) (deprotection step) and step (2) (condensation step), between step (2) and step (3) (oxidation or sulfurization step), and after step (3), a polar solvent (preferably acetonitrile) is added to the reaction solution containing the precipitation accelerator to separate a precipitation mixture containing the 5'-hydroxyl free body obtained in step (1), the phosphite triester compound obtained in step (2) or the resultant oligonucleotide obtained in step (3) (hereinafter, the 5'-hydroxyl free body, the phosphite triester compound and the resultant oligonucleotide are sometimes collectively referred to as the "target product") and the precipitation accelerator from the reaction solution.

[0641] As the polar solvent, the above-mentioned polar solvents can be mentioned. As the polar solvent to be used in this step, acetonitrile is particularly preferred.

[0642] In order to improve the recovery rate of the target product, the amount of the polar solvent (preferably acetonitrile) to be used in this step is preferably not less than 0.5 mL, more preferably not less than 1 mL, further preferably not less than 2 mL, preferably not more than 20 mL, more preferably not more than 10 mL, and further preferably not more than 8 mL, relative to the non-polar solvent contained in each 1 mL of the reaction solution containing the target product.

[0643] When acetonitrile is used, it may contain water to minimize the loss of the target product in the acetonitrile. In this case, the content of water in the acetonitrile is preferably 1-10% (v / v), more preferably 3-8% (v / v). When the water content is too low, the loss of the target product in the acetonitrile will increase, and when the water content is too high, the removal of unnecessary substances entering the acetonitrile will be insufficient.

[0644] Step (6) (Dissolution step)

[0645] In this step, a non-polar solvent is added to the precipitate mixture obtained in step (5) (precipitation and solid-liquid separation step) to prepare a reaction solution. In this step, the preparation cycle including the above steps (1) to (3) can be repeated.

[0646] The explanation of the nonpolar solvent that can be used in this step is the same as that in step (1). Although there is no particular limitation on the concentration of the precipitation mixture in the reaction solution obtained in this step as long as the precipitation mixture is dissolved in the nonpolar solvent, it is preferably 1-30 wt%.

[0647] In the method for producing the oligonucleotide of the present invention, step (5) (precipitation and solid-liquid separation step) and step (6) (dissolution step) are preferably performed after step (3) (oxidation or sulfurization step).

[0648] The method for preparing the oligonucleotide of the present invention may comprise the following steps (7):

[0649] (7) A step of removing all protecting groups of the oligonucleotide to separate the oligonucleotide (hereinafter sometimes referred to as "unprotected oligonucleotide") (deprotection and separation step).

[0650] Step (7) (deprotection and separation step)

[0651] In the preparation method of oligonucleotide of the present invention, according to the type and property of protecting group, preferably deprotection is carried out after step (5), and oligonucleotide is separated. As the method of deprotection, for example, according to the deprotection method described in PROTECTIVE GROUPS INORGANIC SYNTHESIS, the 3rd edition, JOHN WILLY & SONS (1999) etc., the step of removing all protecting groups of oligonucleotide can be carried out. Specifically, the protecting groups of pseudo solid phase protecting groups of the present invention and nucleic acid bases such as phenoxyacetyl, acetyl and cyanoethyl protecting phosphate backbone etc. can be treated with a mixed solution of ammonia, ammonia / ethanol solution or ammonia and methylamine aqueous solution, so as to be completely removed. In addition, the protecting group of 5' hydroxyl can be removed by treating with the acid used in step (1) or the solution after the acid is appropriately diluted. Since oligonucleotide without protecting group is easily degraded by enzyme, it is preferred to separate oligonucleotide under suitable air cleanliness control.

[0652] The progress of the reaction in each step can be determined by a method similar to that of a conventional liquid phase organic synthesis reaction. That is, the reaction can be followed by thin layer silica gel chromatography, high performance liquid chromatography, or the like.

[0653] The 5'-hydroxyl free body, phosphite triester compound or oligonucleotide obtained in step (5) (precipitation and solid-liquid separation step), or the unprotected oligonucleotide obtained in step (7) (deprotection and separation step) can also be further subjected to organic synthesis reaction to produce the desired derivative. The oligonucleotides prepared by the present invention can be used for various purposes, such as various drugs (RNA, DNA, oligonucleic acid drugs, etc.) for humans or animals, foods (functional foods, foods for specific health care), chemical products, macromolecular materials for organisms or industries, etc.

[0654] [Example]

[0655] The present invention is described in more detail below with reference to examples, which should not be construed as limiting the scope of the present invention. Unless otherwise specified, the solvent ratios described below represent volume ratios. Unless otherwise specified, the reagents, equipment and materials used in the present invention are commercially available. In this specification sheet, when represented by abbreviations such as amino acids, each represents an abbreviation based on IUPAC-IUB Commission on Biochemical Nomenclature or a conventional abbreviation in the art.

[0656] Abbreviations of groups and compounds used in Examples and the like are as follows.

[0657] Me:Methyl

[0658] DMTr: 4,4'-dimethoxytrityl

[0659] TBDMS: tert-butyldimethylsilyl

[0660] Boc: tert-Butyloxycarbonyl

[0661] Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

[0662] Trt: trityl

[0663] TOB: 3,4,5-tri(octadecyloxy)benzyl

[0664] DMF: N,N-dimethylformamide

[0665] HBTU: O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0666] Reference Example 1: Continuous synthesis of phosphorthioate dimers in solution

[0667]

[0668] Under argon atmosphere, 5'-O-(4,4'-dimethoxytrityl)deoxythymidine-3'-yl[3,4,5-tri(octadecyloxy)benzyl]succinate (504 mg, 327 μmol) was dissolved in dichloromethane (7.5 mL), pyrrole (213 μL, 3.27 mmol) and trifluoroacetic acid (292 μL, 3.93 mmol) were added and the mixture was stirred at room temperature for 15 min, and the completion of the reaction was confirmed by thin layer chromatography. The reaction mixture was neutralized with pyridine (316 μL, 3.93 mmol) and 1-methylimidazole (156 μL, 1.96 mmol), and then a solution of 4,5-dicyanoimidazole (116 mg, 982 mmol) and 5'-O-(4,4'-dimethoxytrityl)deoxythymidine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (609 mg, 818 μmol) in acetonitrile was added, and the mixture was stirred at room temperature for 60 min, and the completion of the reaction was confirmed by thin layer chromatography. In addition, 3-[(N,N-dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione (184 mg, 898 μmol) was added and the mixture was stirred at room temperature for 30 min. Methanol (100 mL) was added to the reaction solution, and the precipitated solid was filtered using a Kiriyama funnel and then dried to obtain 5'-O-(4,4'-dimethoxytrityl)deoxythymidine-3'-[O-(2-cyanoethyl)]phosphorothionyldeoxythymidine-3'-yl[3,4,5-tri(octadecyloxy)benzyl]succinate (567 mg, yield 91%) as a white solid.

[0669] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, 9H, J = 6.8Hz, Ar-C H3 ),1.28(br,s,90H,(C H2 ) 15 ),1.30(s,3H,N1 5 -C H3 ),1.46(s,3H,N2 5 -C H3 ),1.73(m,6H,Ar-OCH2C H2 ),2.28(m,2H,2'1- H ),2.41(m,2H,2'2- H ),2.68(m,2H,5'1- H and m,4H,succinyl-C H2 C H2 ),3.44(m,2H,5'2- H),3.79(s,6H,DMTr-OC H3 ),3.95(m,6H,Ar-OC H2 ),4.10(m,1H,4'1- H ),4.31(m,4H,cyanoethyl-C H2 C H2 and m,1H,4'2- H ),5.01(s,2H,Ar-CH2-succinyl),5.26(m,1H,3'1- H ),5.32(m,1H,3'2- H ),6.27(m,1H,1'1- H ),6.38(m,1H,1'2- H ),6.53(s,2H,Ar- H ),6.84-7.29(m,13H,DMTr-Ar- H ),7.29(m,1H,N1 6 - H ),7.56(m,1H,N2 6 - H )

[0670] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, 9H, Ar-CH 3 ),1.28(br,s,90H,(C H2 ) 15 ),1.30(s,3H,N1 5 -C H3 ),1.46(s,3H,N2 5 -C H3 ),1.73(m,6H,Ar-OCH2C H2 ),2.41(m,2H,2'2- H ),2.68(m,2H,2'1- H and m,4H,succinyl-C H2 C H2 ),2.77(m,2H,5'1- H ),3.44(m,2H,5'2- H ),3.79(s,6H,DMTr-OC H3 ),3.95(m,6H,Ar-OC H2 ),4.17(m,1H,4'1- H ),4.31(m,4H,cyanoethyl-C H2 C H2 andm,1H,4'2-H ),5.01(s,2H,Ar-C H2 -succinyl), 5.33(m,1H,3'1- H and m,1H,3'2- H ),6.27(m,1H,1'1- H ),6.38(m,1H,1'2- H ),6.53(s,2H,Ar- H ),6.84-7.29(m,13H,DMTr-Ar- H ),7.29(m,1H,N1 6 - H ),7.56(m,1H,N2 6 - H )

[0671] Reference Example 2: Continuous Synthesis of Phosphorothioate Pentamers in Solution

[0672] The operation similar to that of Reference Example 1 was repeated 4 times to obtain 5′-O-(4,4-dimethoxytrityl)-N 2 -isobutyryl-deoxyguanosine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 6 -benzoyl-deoxyadenosine 3'-[O-(2-cyanoethyl)]phosphorothioyl-deoxythymidine 3'-[O-(2-cyanoethyl)]phosphorothioyl-deoxythymidine-3'-yl[3,4,5-tri(octadecyloxy)benzyl]succinate (1.94 g).

[0673] Reference Example 3: Continuous Synthesis of Phosphorothioate Decamers in Solution

[0674] The similar operation to that of Reference Example 1 was repeated 9 times to obtain 5′-O-(4,4′-dimethoxytrityl)-N 2 -isobutyryl-deoxyguanosine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 6 -Benzoyl-deoxyadenosine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 6 -Benzoyl-deoxyadenosine 3'-[O-(2-cyanoethyl)]phosphorothioate-deoxythymidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 2 -isobutyryl-deoxyguanosine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N6 -benzoyl-deoxyadenosine 3'-[O-(2-cyanoethyl)]phosphorothioyl-deoxythymidine 3'-[O-(2-cyanoethyl)]phosphorothioyl-deoxythymidine 3'-yl 3,4,5-tri(octadecyloxy)benzyl succinate (1.41 g).

[0675] Reference Example 4: Continuous synthesis of phosphorothioate 20-mers in solution

[0676] The operation similar to that of Reference Example 1 was repeated 19 times to obtain 5'-O-(4,4'-dimethoxytrityl)-deoxythymidine 3'-[O-(2-cyanoethyl)]thiophosphoryl-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 2 -isobutyryl-deoxyguanosine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-deoxythymidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 2 -isobutyryl-deoxyguanosine 3'-[O-(2-cyanoethyl)]phosphorothioate-deoxythymidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 2 -isobutyryl-deoxyguanosine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 6 -Benzoyl-deoxyadenosine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 6 -Benzoyl-deoxyadenosine 3'-[O-(2-cyanoethyl)]phosphorothioate-deoxythymidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 2 -isobutyryl-deoxyguanosine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 6 -benzoyl-deoxyadenosine 3'-[O-(2-cyanoethyl)]phosphorothioyl-deoxythymidine 3'-[O-(2-cyanoethyl)]phosphorothioyl-deoxythymidine 3'-[O-(2-cyanoethyl)]phosphorothioyl-deoxythymidine 3'-yl 3,4,5-tri(octadecyloxy)benzyl succinate (1.61 g).

[0677] Comparative Example 1: Synthesis of methyl 3,4,5-tris(16-methylheptadecan-1-yloxy)benzoate

[0678] (1) Synthesis of 16-methylheptadecan-1-yl bromide

[0679]

[0680] 16-Methylheptadecan-1-yl alcohol (5.00 g, 18.5 mmol) was dissolved in hydrobromic acid (50 mL), concentrated sulfuric acid (84 μL, 1.59 mmol) was added, and the mixture was stirred at 100 ° C for 16 hr. The reaction was confirmed to be complete by thin layer chromatography, and then the reaction solution was cooled to room temperature and hexane (150 mL) was added. The reaction solution was washed twice with saturated sodium bicarbonate aqueous solution (100 mL) and once with saturated brine (50 mL). The organic layer was then dried with magnesium sulfate and purified by short silica gel column chromatography (hexane) to obtain the target compound (5.56 g, yield 90%).

[0681] (2) Synthesis of methyl 3,4,5-tris(16-methylheptadecan-1-yloxy)benzoate

[0682]

[0683] Methyl 3,4,5-trihydroxybenzoate (1.03 g, 5.57 mmol), 16-methyl-heptadecylbromide (5.56 g, 16.7 mmol), potassium carbonate (3.85 g, 27.9 mmol) were dissolved in N,N-dimethylformamide (50.0 mL), and the mixture was stirred at 110 ° C for 16 hours. After the reaction was confirmed to be complete by thin layer chromatography, the reaction solution was cooled to room temperature and ethyl acetate (200 mL) was added. The organic layer was washed twice with water (100 mL), once with saturated sodium bicarbonate aqueous solution (100 mL), and once with saturated brine (100 mL), then dried with sodium sulfate and concentrated under reduced pressure to obtain the target compound (4.66 g, yield 88%).

[0684] 1 H-NMR (400MHz, CDCl3): δ=0.83-0.90(m,18H,-C H3 ),1.05-1.51(m,81H,-O-CH2-CH2-(C H2 ) 13 -C H (CH3)2),1.70-1.84(m,6H,-O-CH2-C H2 -(CH2) 13 -CH(CH3)2),3.88(s,3H,C(O)OC H3),3.99-4.02(m,6H,Ar-OC H2 ),7.25(s,2H,Ar- H )

[0685] Comparative Example 2: Synthesis of methyl 4-octadecyloxybenzoate

[0686]

[0687] Under argon atmosphere, potassium carbonate (2.72g, 19.7mmol), methyl 4-hydroxybenzoate (1.00g, 6.57mmol), 1-bromooctadecane (2.63g, 7.88mmol) were dissolved in N,N-dimethylformamide (60mL), and the mixture was stirred at 90°C for 16hr. After the reaction was confirmed to be complete by thin layer chromatography, ethyl acetate (100mL) and tetrahydrofuran (20mL) were added to the reaction solution, and the organic layer was washed 3 times with water (100mL) and once with saturated brine (100mL). The organic layer was then evaporated under reduced pressure. Ethyl acetate (50mL) was added to the concentrated residue, and the mixture was dissolved by heating to 70°C and then cooled to 5°C to obtain the target compound (2.61g, yield 98%).

[0688] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, 3H, J = 6.8Hz, -CH2CH2 (CH2) 15 C H3 ),1.26-1.49(m,30H,-CH2CH2(C H2 ) 15 CH3), 1.76-1.83(m,2H,-CH2C H2 (CH2) 15 CH3),3.88(s,3H,-C(O)OC H3 ),4.00(t,2H,J=6.4Hz,-C H2 CH2(CH2) 15 CH3),6.90(d,2H,J=9.2Hz,Ar-H),7.98(d,2H,J=9.2Hz,Ar-H)

[0689] Example 1: Synthesis of methyl 3,5-bis(octadecyloxy)benzoate

[0690]

[0691] Under argon atmosphere, potassium carbonate (19.9 g, 144 mmol), methyl 3,5-dihydroxybenzoate (3.60 g, 21.4 mmol), 1-bromooctadecane (15.5 g, 46.7 mmol) were dissolved in N,N-dimethylformamide (200 mL), and the mixture was stirred at 70 ° C for 16 hours. After the reaction was confirmed to be complete by thin layer chromatography, the reaction solution was poured into water (800 mL) for precipitation to obtain a solid. The obtained solid was dissolved in dichloromethane (200 mL), and after the solid was precipitated by methanol (400 mL), it was collected by filtration and dried to obtain the target compound (13.9 g, yield 97%).

[0692] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, 6H, J = 6.6Hz, -OCH2CH2 (CH2) 15 C H3 ),1.25-1.55(m,76H,-OCH2CH2(C H2 ) 15 CH3),1.77(m,4H,-OCH2C H2 (CH2) 15 CH3),3.89(s,3H,C(O)OC H3 ),3.96(t,4H,J=6.6Hz,-OC H2 CH2(CH2) 15 CH3),6.63(s,1H,Ar-H),7.15(s,2H,Ar-H)

[0693] Example 2: Synthesis of methyl 3,5-bis(docosyloxy)benzoate

[0694]

[0695] Under argon atmosphere, potassium carbonate (20.0 g, 145 mmol), methyl 3,5-dihydroxybenzoate (3.60 g, 21.4 mmol), 1-bromodocosane (17.9 g, 46.0 mmol) were dissolved in N,N-dimethylformamide (200 mL), and the mixture was stirred at 70 ° C for 16 hours. After confirming the completion of the reaction by thin layer chromatography, the reaction solution was poured into water (1.00 L) for precipitation to obtain a solid. The obtained solid was dissolved in dichloromethane (500 mL), and after the solid was precipitated by methanol (400 mL), it was collected by filtration and dried to obtain the target compound (16.1 g, yield 96%).

[0696] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, 6H, J = 6.6Hz, -CH3 ),1.25-1.55(m,76H,-OCH2CH2(C H2 ) 19 CH3),1.77(m,4H,-OCH2C H2 (CH2) 19 CH3),3.89(s,3H,C(O)OC H3 ),3.96(t,4H,J=6.6Hz,-OC H2 CH2(CH2) 19 CH3),6.63(s,1H,Ar-H),7.15(s,2H,Ar-H)

[0697] Example 3: 2,3,4-Tris(octadecyloxy)benzophenone

[0698]

[0699] Under argon atmosphere, potassium carbonate (4.35 g, 31.5 mmol), 2,3,4-trihydroxybenzophenone (1.61 g, 7.00 mmol), 1-bromooctadecane (7.32 g, 22.0 mmol) were dissolved in N,N-dimethylformamide (30 mL), and the mixture was stirred at 80 ° C for 16 hours. After the reaction was confirmed to be complete by thin layer chromatography, the reaction solution was poured into water (200 mL) for precipitation to obtain a solid. The obtained solid was dissolved in dichloromethane (200 mL), and after the solid was precipitated by methanol (300 mL), it was collected by filtration and dried to obtain the target compound (6.31 g, yield 91%).

[0700] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, 9H, J = 6.7Hz, -C H3 ),1.11-1.55(m,90H,-O-CH2CH2(C H2 ) 15 CH3), 1.74-1.87(m,4H,-O-CH2C H2 (CH2) 15 CH3), 3.88(t, 2H, J = 6.6 Hz, -OC H2 CH2(CH2) 15 CH3),3.99(t,2H,J=6.6Hz,-OC H2 CH2(CH2) 15 CH3), 4.02(t, 2H, J = 6.6 Hz, -OC H2 CH2(CH2) 15CH3),6.69(d,1H,J=8.6Hz,Ar-H),7.12(d,1H,J=8.6Hz,Ar-H),7.41(m,2H,Ar-H),7.53(m,1H,Ar-H),7.77(m,2H,Ar-H)

[0701] Example 4: Synthesis of 3,4,5-tri(octadecyloxy)toluene

[0702]

[0703] Under hydrogen atmosphere, 2-tri(octadecyloxy)benzyloxy-4-methoxy-benzaldehyde oxime (487mg, 470μmol) was dissolved in a mixed solvent of THF (40mL) and methanol (10mL), and after adding 10wt% Pd / C (53.3mg), the mixture was stirred at room temperature for 64hr. After confirming the completion of the reaction by thin layer chromatography, the reaction solution was filtered through diatomaceous earth, and acetonitrile (200mL) was added to the filtrate to precipitate the solid. The solid was collected by filtration and dried to obtain the target compound (391mg, yield 93%).

[0704] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, 9H, J = 6.6Hz, -OCH2CH2 (CH2) 19 C H3 ),1.26-1.80(m,96H,-OCH2C H2 (C H2 ) 19 CH3),2.27(s,3H,Ar-Me),3.89-3.96(m,6H,-OC H2 CH2(CH2) 19 CH3),6.35(s,2H,Ar-H)

[0705] Example 5: Synthesis of methyl 3,4,5-tris[3,4,5-tri(octadecyloxy)benzyloxy]benzoate

[0706] Under an argon atmosphere, potassium carbonate (1.39 g, 10.1 mmol), methyl 3,5-hydroxybenzoate (181 mg, 1.00 mmol), and 3,4,5-tri(octadecyloxy)benzyl chloride (2.81 g, 3.01 mmol) were dissolved in N,N-dimethylformamide (20 mL), and the mixture was stirred at 70 ° C for 18 hr. After the reaction was confirmed to be complete by thin layer chromatography, the reaction solution was poured into water (50 mL) for precipitation to obtain a solid. The obtained solid was dissolved in dichloromethane (50 mL), and after the solid was precipitated by acetonitrile (200 mL), it was collected by filtration and dried to obtain a crude product of the target product. The obtained crude product was purified by silica gel column chromatography (dichloromethane / hexane (volume ratio) = 1 / 2-1 / 1) to obtain the target compound (2.31 g, 81%).

[0707] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, 9H, J = 7.0Hz, -O (CH2) 17 C H3 ),1.25-1.74(m,291H,-OCH2(C H2 +N 5 -C H3 ) 16 CH3),3.75(m,6H,-OC H2 (CH2) 16 CH3),3.90(s,3H,-C(O)OC H3 ),3.86-3.94(2m,12H,-OC H2 (CH2) 16 CH3),5.02(s,6H,3×- H2C-Ar ),6.59(s,2H,Ar- H ),6.62(s,4H,Ar- H ),7.38(s,2H,Ar-H)

[0708] Example 6: Synthesis of methyl 3,4,5-tri(octadecyloxy)benzoate

[0709]

[0710] 1-Bromooctadecane (64.0 g, 192 mmol), potassium carbonate (79.6 g, 576 mmol), methyl 3,4,5-trihydroxybenzoate (11.0 g, 60.0 mmol) were dissolved in N,N-dimethylformamide (600 mL), and the mixture was stirred at 90 ° C for 16 hours. After confirming the completion of the reaction by thin layer chromatography (hexane / dichloromethane (volume ratio) = 2 / 1 and dichloromethane / methanol (volume ratio) = 9 / 1), the reaction solution was poured into water (2.00 L) to precipitate the solid, which was then filtered and dried. The dried solid was dissolved in dichloromethane (500 mL), and acetonitrile (1.50 L) was added to precipitate the solid. The solid was collected by filtration and dried to obtain the target compound (56.7 g, 100%).

[0711] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, 9H, J = 6.8Hz, -OCH2 (CH2) 16 C H3 ),1.21-1.51(m,90H,-O-CH2CH2(C H2) 15 CH3),1.70-,6H,-O-CH2-C H2- (CH2 ) 15 -CH3),3.89(s,3H,C(O)OCH 3 ),3.99-4.03(m,6H,Ar-OC H2 ),4.99(s,2H,Ar-H),7.25(s,2H,Ar-H)

[0712] Example 7: Synthesis of 3,4,5-tri(octadecyloxy)benzyl acetate

[0713] (1) Synthesis of 3,4,5-tri(octadecyloxy)benzyl alcohol

[0714]

[0715] Under argon atmosphere, methyl 3,4,5-tri(octadecyloxy)benzoate (10.0 g, 10.6 mmol) was dissolved in tetrahydrofuran (150 mL), lithium aluminum hydride (810 mg, 21.3 mmol) was added, and the mixture was stirred at room temperature for 30 min. After the reaction was confirmed to be complete by thin layer chromatography, 4M aqueous sodium hydroxide solution (6.0 mL) was quenched and the reaction solution was filtered. Methanol (500 mL) was added to the filtrate to precipitate a solid, which was collected by filtration and dried to obtain the target compound (9.65 g, yield 99%).

[0716] (2) Synthesis of 3,4,5-tri(octadecyloxy)benzyl acetate

[0717]

[0718] 3,4,5-tri(octadecyloxy)benzyl alcohol (10.0 g, 10.5 mmol), acetic anhydride (9.90 mL, 105 mmol), pyridine (8.40 mL, 105 mmol), 4-dimethylaminopyridine (128 mg, 1.05 mmol) were dissolved in dichloromethane (100 mL), and the mixture was stirred at room temperature for 5 min. After the reaction was confirmed to be complete by thin layer chromatography, acetonitrile (500 mL) was added to precipitate a solid, which was collected by filtration. The obtained solid was purified by silica gel column chromatography (hexane / dichloromethane = 2 / 1-1 / 1) to quantitatively obtain the target compound (10.4 g) as a white solid.

[0719] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, 9H, J = 6.8Hz, -C H3 ),1.26-1.50(m,90H,-O-CH2-CH2-(C H2) 15 CH3), 1.70-1.83(m,6H,-O-CH2-C H2 -(CH2) 15 -CH3),2.10(s,3H,C(O)C H3 ),3.92-3.98(m,6H,Ar-OC H2 ),4.99(s,2H,Ar-C H2 ),6.54(s,2H,Ar-H)

[0720] Example 8: Synthesis of 1,2,3-tri(octadecyloxy)propane

[0721]

[0722] Under argon atmosphere, potassium carbonate (12.4 g, 90.0 mmol), glycerol (921 mg, 10.0 mmol), 1-bromooctadecane (11.0 g, 33.0 mmol) were dissolved in N,N-dimethylformamide (100 mL), and the mixture was stirred at 110 ° C for 16 hours. After confirming the completion of the reaction by thin layer chromatography, the reaction solution was poured into water (400 mL) for precipitation to obtain a solid. The obtained solid was purified by silica gel column chromatography (dichloromethane) to obtain the target compound (4.30 g, 51%).

[0723] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, 9H, J = 6.8Hz, -CH2 (CH2) 16 C H3),1.18-1.36(m,95H,-CH2CH2(C H2 ) 15 CH3, H C(C H2 )2),1.53-1.62(m,6H,-CH2C H2 (CH2) 15 CH3), 3.62-3.66(m,6H,-C H2 (CH2) 16 CH3)

[0724] Example 9: Synthesis of 1,3-di(octadecyloxy)-2,2-di(octadecyloxymethyl)propane

[0725]

[0726] Under argon atmosphere, potassium carbonate (6.10 g, 44.1 mmol), pentaerythritol (501 mg, 3.67 mmol), 1-bromooctadecane (5.50 g, 16.5 mmol) were dissolved in N, N-dimethylformamide (35 mL), and the mixture was stirred at 110 ° C for 16 hours. After the reaction was confirmed to be complete by thin layer chromatography, the reaction solution was poured into water (400 mL) for precipitation to obtain a solid. The obtained solid was dissolved in dichloromethane (200 mL), and after the solid was precipitated by methanol (300 mL), it was collected by filtration and dried to obtain the target compound (3.57 g, 85%).

[0727] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, J = 6.8Hz, 12H, -CH2 (CH2) 16 C H3 ),1.17-1.36(m,128H,-CH2CH2(C H2) 16 CH3),1.53-1.60(m,8H,-C(C H2)4 ),3.62-3.66(m,8H,-C H2 (CH2) 16 CH3)

[0728] Example 10: Synthesis of methyl 4-docosyloxybenzoate

[0729]

[0730] Under argon atmosphere, potassium carbonate (1.36 g, 9.85 mmol), methyl 4-hydroxybenzoate (500 mg, 3.29 mmol), 1-bromooctadecane (1.28 g, 3.62 mmol) were dissolved in N, N-dimethylformamide (30 mL), and the mixture was stirred at 90 ° C for 16 hours. After confirming the completion of the reaction by thin layer chromatography, the reaction solution was poured into water (400 mL) for precipitation to obtain a solid. After the obtained solid was dissolved in ethyl acetate (50 mL) at 70 ° C, it was cooled to room temperature to precipitate crystals, which were then collected by filtration and dried to obtain the target compound (1.37 g, 91%).

[0731] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, 3H, J = 6.8Hz, -CH2CH2 (CH2) 15 C H3 ),1.26-1.58(m,38H,-CH2CH2(C H2) 19 CH3), 1.76-1.83(m,2H,-CH2C H2 (CH2) 19 CH3),3.88(s,3H,-C(O)OC H3 ),4.00(t,2H,J=6.4Hz,-C H2 CH2(CH2) 15 CH3),6.90(d,2H,J=9.5Hz,Ar-H),7.98(d,2H,J=9.5Hz,Ar-H)

[0732] Example 11: Synthesis of methyl 3,4,5-tri(octadecyloxy)cyclohexanecarboxylate

[0733]

[0734] Cyclohexane is added to 3,4,5-tri(octadecyloxy)benzoic acid methyl ester (1.00g, 10.6mmol), 5% rhodium-carbon (0.8g) is added, and hydrogen reduction is carried out at 80°C and 10atm for 16hr. Tetrahydrofuran (10mL) is added to the mixture after hydrogen reduction and the catalyst is filtered off. The filtrate is concentrated, methanol (8mL) is added to the concentrated solution, and the mixture is stirred. The obtained precipitate is collected by filtration, and dried to obtain the target compound (820mg, yield 82%).

[0735] 1 H-NMR (400MHz, CDCl3): δ = 0.89 (9H, t, J = 6.9Hz, C 17 H 34-Me),1.25-1.44(90H,m),1.52-1.60(6H,m),1.86-1.94(4H,m),2.23(1H ,m),3.09-3.14(2H,m),3,41-3.47(4H,m),3.64-3.68(5H,m),3.86(1H,s)

[0736] Example 12: Synthesis of methyl 2,4-di(docosyloxy)benzoate

[0737]

[0738] Under argon atmosphere, potassium carbonate (29.9 g, 217 mmol), methyl 2,4-dihydroxybenzoate (3.65 g, 21.7 mmol), 1-bromodocosane (17.33 g, 44.5 mmol) were dissolved in N,N-dimethylformamide (150 mL), and the mixture was stirred at 70 ° C for 16 hours. After the reaction was confirmed to be complete by thin layer chromatography, the reaction solution was poured into water (1.00 L) for precipitation to obtain a solid. The obtained solid was dissolved in dichloromethane (500 mL), and after the solid was precipitated by methanol (400 mL), it was collected by filtration and dried to obtain the target compound (16.7 g, yield 98%).

[0739] 1 H-NMR (400MHz, CDCl3): δ = 0.88 (t, 6H, J = 6.6Hz, -C H3 ),1.13-1.56(m,76H,-OCH2CH2(C H2 ) 19 CH3),1.81(m,4H,-OCH2C H2 (CH2) 19 CH3),3.89(s,3H,C(O)OC H3 ),4.02(dd,4H,J=6.6,14.8Hz,-OC H2 CH2(CH2) 19 CH3),6.63(s,1H,Ar-H),7.15(s,2H,Ar-H)

[0740] Example 13: Synthesis of pivaloyloxymethyl-3,4,5-tri(octadecyloxy)benzene

[0741]

[0742] Under argon atmosphere, 3,4,5-tri(octadecyloxy)benzyl alcohol (2.73 g, 2.99 mmol) was dissolved in dichloromethane (25 mL), and triethylamine (1.67 mL, 12.0 mmol), pivaloyl chloride (781 μL, 6.00 mmol), 4-dimethylaminopyridine (7.32 mg, 600 μmol) were added in sequence, and the mixture was stirred at room temperature for 7 hr. The reaction was confirmed to be complete by thin layer chromatography, and acetonitrile (125 mL) was added to the reaction solution to precipitate a solid, which was collected by filtration. The obtained solid was dried under reduced pressure to quantitatively obtain the target compound (2.80 g) as a white solid.

[0743] 1 H-NMR (400MHz, CDCl3): δ = 0.879 (t, J = 7.06Hz, 9H), 1.23 (s, 9H), 1.24-1.40 (m, 84H), 1.40-1.52 ( m,6H),1.70-1.81(m,6H),3.92(t,J=6.8Hz,2H),3.95(t,J=6.8Hz,4H),5.01(s,2H),6.51(s,2H)

[0744] Example 14: (2S)-2-(Acetylamino)-4-methylpentanoyloxymethyl-3,4,5-tri(octadecyloxy) Synthesis of 2-(4-(2-amino)benzene

[0745]

[0746] 3,4,5-tri(octadecyloxy)benzyl alcohol (1.83 g, 2.00 mmol), N-acetyl leucine (700 mg, 4.09 mmol), 2,6-dimethylaminopyridine (2.58 mg, 20.0 μmol) were dissolved in dichloromethane (20 mL), and then diisopropylethylamine (1.16 mL, 6.79 mmol) and HBTU (1.90 g, 5.00 mmol) were added, and the mixture was stirred at room temperature for 15 hr, and then further stirred at 40 ° C for 21 hr. After filtering the reaction solution, acetonitrile (100 mL) was added to the filtrate to precipitate a solid, which was collected by filtration. The obtained solid was dried under reduced pressure to obtain the target compound (1.97 g) as a white solid.

[0747] 1H-NMR (400MHz, CDCl3): δ=0.879 (t, J=7.06Hz, 9H), 0.913 (d, J=3.2Hz, 3H), 0.92 9(d,J=3.2Hz,3H),1.22-1.40(m,84H),1.40-1.52(m,7H),2.02(s,3H),1.60-1. 82(m,8H),3.94(t,J=6.4Hz,2H),3.95(t,J=6.8Hz,4H),4.684(ddd,J=13.6,4.8 ,4.8Hz,1H),5.024(d,12.4Hz),5.082(d,12.4Hz),5.817(d,8.4Hz),6.52(s,2H)

[0748] Example 15: Synthesis of triisopropylsilyloxymethyl-3,4,5-tri(octadecyloxy)benzene

[0749]

[0750] Under argon atmosphere, 3,4,5-tri(octadecyloxy)benzyl alcohol (4.57 g, 5.00 mmol) and imidazole (1.36 g, 20.0 mmol) were dissolved in a mixed solvent of dichloromethane (40 mL) and N,N-dimethylformamide (10 mL), triisopropylsilyl chloride (2.12 mL, 10.0 mmol) was added and the mixture was stirred at 40 ° C for 6 hr. After cooling to room temperature, acetonitrile (250 mL) was added to the reaction solution to precipitate a solid, which was collected by filtration. The obtained solid was dried under reduced pressure to obtain the target compound (5.23 g) as a white solid.

[0751] 1 H-NMR (400MHz, CDCl3): δ = 0.879 (t, J = 7.06Hz, 9H), 1.09 (d, 18H, 6.0Hz), 1.12-1.20 (m, 3H), 1.22-1.40 (m, 84H ),1.40-1.52(m,6H),1.70-1.82(m,6H),3.91(t,J=6.8Hz,2H),3.97(t,J=6.8Hz,4H),4.75(s,2H),6.56(s,2H)

[0752] Example 16: Synthesis of 4-(3,4,5-tri(octadecyloxy)benzoyl)-morpholine

[0753]

[0754] Under argon atmosphere, 3,4,5-tri(octadecyloxy)benzoic acid (5.00 g, 5.39 mmol) was dissolved in dichloromethane (50 mL), and then HBTU (6.13 g, 16.2 mmol), morpholine (1.31 mL, 16.2 mmol) and diisopropylethylamine (2.90 mL, 16.2 mmol) were added in sequence, and the mixture was stirred at room temperature for 18 hr. After confirming the end point of the reaction by thin layer chromatography (hexane / dichloromethane / ethyl acetate = 25 / 50 / 25), acetonitrile (250 mL) was added to the reaction solution to precipitate a solid, which was collected by filtration. The obtained solid was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0-95 / 5) to obtain the target compound (5.18 g) as a white solid.

[0755] 1 H-NMR (400MHz, CDCl3): δ = 0.879 (t, J = 6.8Hz, 9H), 1.26-1.47 (m, 84H), 1.42-1.53 ​​(m, 6H), 1.71-1.80 (m, 6H), 3.69 (m, 8H), 3.95 (m, 6H), 6.51 (s, 2H)

[0756] Test Example 1

[0757] As the precipitate, the pentamer synthesized in Reference Example 2 or the 20-mer synthesized in Reference Example 4 was used to study the relationship between the amount of precipitation promoter added and the recovery rate of the precipitate. The precipitate shown in the amounts of Tables 1 and 2 and the precipitation promoter (methyl 3,4,5-tri(octadecyloxy)benzoate) synthesized in Example 6 were dissolved in dichloromethane (2 mL), and then the solid was precipitated with the amount of acetonitrile shown in Tables 1 and 2, and the obtained solid was collected by filtration using a Kiriyama funnel (21 m / m) and filter paper (No-5A), and the solid was washed with acetonitrile (20 mL) and dried under reduced pressure. The recovery rate calculated from the amount of the precipitate used (i.e., the pentamer of Reference Example 2 or the 20-mer of Reference Example 4) and the amount of the precipitation promoter used, and the amount of the recovered mixture of the dried precipitate and the precipitation promoter (=100x(amount of the recovered mixture of the dried precipitate and the precipitation promoter (mg)) / (amount of the precipitate used (mg) + amount of the precipitation promoter used (mg))) is shown in Tables 1 and 2.

[0758] Table 1

[0759]

[0760] *1 Molar equivalent of the precipitation promoter of Example 6 relative to the pentamer of Reference Example 2

[0761] Table 2

[0762]

[0763] *1 The molar equivalent of the precipitation promoter of Example 6 relative to the 20-mer of Reference Example 4

[0764] Test Example 2

[0765] As the precipitate, the decamer synthesized in Reference Example 3 was used to study the relationship between the type of precipitation promoter and the recovery rate of the precipitate. The decamer of Reference Example 3 and the precipitation promoter synthesized in any of Comparative Examples 1 and 2 and Examples 1-12 in the amounts shown in Table 3 were dissolved in dichloromethane (0.5 mL), and then the solid was precipitated with acetonitrile (2.5 mL). The obtained solid was collected by filtration using a Kiriyama funnel (21 m / m) and filter paper (No-5A), and the solid was washed with acetonitrile (2.0 mL) and dried under reduced pressure. The recovery rate (=100x (the amount of the mixture of the recovered dried precipitate and precipitation promoter (mg)) / (the amount of the used precipitate (mg) + the amount of the used precipitation promoter (mg))) calculated from the amount of the used precipitate (i.e., the decamer of Reference Example 3) and the amount of the used precipitation promoter, as well as the amount of the recovered dried precipitate and precipitation promoter is shown in Table 3.

[0766] Table 3

[0767]

[0768] *1 Molar equivalent of each precipitation promoter relative to the decamer of Reference Example 3

[0769] As shown in Table 3, the precipitation accelerators of Examples 1 to 12 can promote the precipitation of the decamer of Reference Example 3 and improve its recovery rate. On the other hand, when the precipitation accelerator of Comparative Example 1 having a branched aliphatic hydrocarbon group and the precipitation accelerator of Comparative Example 2 having a straight aliphatic hydrocarbon group but the total number of carbon atoms of the group is less than 20 are used, the recovery rate of the decamer of Reference Example 3 is reduced compared to the case where these precipitation accelerators are not used.

[0770] Test Example 3

[0771] As the precipitate, the pentamer synthesized in Reference Example 2 was used to study the relationship between the type of precipitation promoter and the recovery rate of the precipitate. The pentamer of Reference Example 2 and the precipitation promoter synthesized in any one of Examples 13-15 in the amounts shown in Table 4 were dissolved in dichloromethane. The amount of acetonitrile shown in Table 4 was added dropwise to the resulting solution under stirring at 24°C for 3 minutes to precipitate the solid to obtain a slurry. The resulting slurry was allowed to stand in an ice bath for 10 minutes, and the precipitated solid was collected by filtration using a 25Φ Kiriyama funnel and 5C filter paper, and the resulting solid was washed with the amount of acetonitrile shown in Table 4. The resulting solid was dried overnight under reduced pressure. The recovery rate (=100x(amount of the recovered dried mixture of the precipitate and the precipitation promoter (mg)) / (amount of the precipitate used (mg) + amount of the precipitation promoter used (mg))) calculated from the amount of the precipitate used (i.e., the pentamer of Reference Example 2) and the amount of the precipitation promoter used, as well as the amount of the recovered dried mixture of the precipitate and the precipitation promoter is shown in Table 4.

[0772] Table 4

[0773]

[0774] *1 Molar equivalent of each precipitation promoter relative to the pentamer of Reference Example 2

[0775] Test Example 4

[0776] As the precipitate, the twenty-mer synthesized in Reference Example 4 was used to study the relationship between the type of precipitation promoter and the recovery rate of the precipitate. The pentamer of Reference Example 2 and the precipitation promoter synthesized in any one of Examples 13-15 in the amounts shown in Table 5 were dissolved in dichloromethane. Acetonitrile (10.5 mL) was added dropwise to the resulting solution under stirring for 3 min at 24°C to precipitate the solid to obtain a slurry. The resulting slurry was allowed to stand in an ice bath for 10 min, and the precipitated solid was collected by filtration using a 25Φ Kiriyama funnel and 5C filter paper, and the resulting solid was washed with acetonitrile (5.0 mL). The resulting solid was dried overnight under reduced pressure. The recovery rate (=100x(amount of the recovered dried mixture of the precipitate and the precipitation promoter (mg)) / (amount of the precipitate used (mg) + amount of the precipitation promoter used (mg))) calculated from the amount of the precipitate used (i.e., the twenty-mer of Reference Example 4) and the precipitation promoter used, as well as the amount of the recovered dried mixture of the precipitate and the precipitation promoter is shown in Table 5.

[0777] Table 5

[0778]

[0779] *1Molar equivalent of each precipitation promoter relative to the 20-mer of Reference Example 4

[0780] Test Example 5

[0781] First, a peptide recovery test was performed without using a precipitation accelerator. Specifically, a peptide (H-Lys(Boc)-Pro-Pro-Ala-Lys(Boc)-Leu-Gln(Trt)-Pro-Arg(Pbf)-OTOB, 435 mg, 0.166 mmol) in which the carboxyl group was protected by 3,4,5-tri(octadecyloxy)benzyl (TOB) was dissolved in chloroform (10 mL). The resulting solution was concentrated under reduced pressure, and about 5 mL of chloroform was removed from the solution to obtain a concentrate. Acetonitrile (20 mL) was added to the concentrate to precipitate a solid. The precipitate was collected by filtration and dried under reduced pressure to obtain a dried peptide. The recovery rate calculated from the amount of peptide used and the amount of dried peptide recovered (=100x(amount of dried peptide recovered (mg)) / (amount of peptide used (mg))) was 71%.

[0782] Next, a peptide recovery test was performed in the same manner as above except that 3,4,5-tri(octadecyloxy)benzyl alcohol was used as a precipitation accelerator. Specifically, a peptide (H-Lys(Boc)-Pro-Pro-Ala-Lys(Boc)-Leu-Gln(Trt)-Pro-Arg(Pbf)-OTOB, 435 mg, 0.166 mmol) in which the carboxyl group was protected by 3,4,5-tri(octadecyloxy)benzyl (TOB) and 3,4,5-tri(octadecyloxy)benzyl alcohol (200 mg, 0.22 mmol) were dissolved in chloroform (10 mL). The resulting solution was concentrated under reduced pressure, and about 5 mL of chloroform was removed from the solution to obtain a concentrate. Acetonitrile (20 mL) was added to the concentrate to precipitate a solid. The precipitate was collected by filtration and dried under reduced pressure to obtain a mixture of a dried peptide and a precipitation accelerator. The recovery rate calculated from the amount of peptide used, the amount of precipitation promoter used, and the amount of the recovered mixture of dried peptide and precipitation promoter (=100x(amount of the recovered mixture of dried peptide and precipitation promoter (mg)) / (amount of peptide used (mg) + amount of precipitation promoter used (mg))) is 97%.

[0783] Example 17: Continuous Synthesis of Phosphorothioate Dimers in Solution by Using a Precipitation Accelerator

[0784] Under an argon atmosphere, 5'-O-(4,4'-dimethoxytrityl)deoxythymidine-3'-yl[3,4,5-tri(octadecyloxy)benzyl]succinate (1.85 g, 1.2 mmol) and 3,4,5-tri(octadecyloxy)benzyl acetate (1.86 g, 1.20 mmol, which is the precipitation promoter synthesized in Example 7) were dissolved in dichloromethane (40 mL), indole (1.40 g, 12.0 mmol) and trifluoroacetic acid (88.9 μL, 1.20 mmol) were added, and the mixture was stirred at room temperature for 2 hr. The completion of the reaction was confirmed by thin layer chromatography. After neutralizing the reaction mixture with 1-methylimidazole (99.7 μL, 1.26 mmol), 4,5-dicyanoimidazole (476 mg, 4.03 mmol), 5'-O-(4,4'-dimethoxytrityl)deoxythymidine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (2.00 g, 2.69 mmol) in acetonitrile were added, and the mixture was stirred at room temperature for 60 min, and completion of the reaction was confirmed by thin layer chromatography. In addition, 3-[(N,N-dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione (608 mg, 2.96 mmol) was added and the mixture was stirred at room temperature for 30 min. Then, acetic anhydride (56.6 μL, 0.60 mmol), 2,4,6-trimethylpyridine (79.1 μL, 0.60 mmol), and 1-methylimidazole (47.5 μL, 0.60 mmol) were added and the mixture was stirred at room temperature for 15 min. Acetonitrile (200 mL) was added to the reaction solution, and the precipitated solid was filtered using a Kiriyama funnel and then dried to obtain 5'-O-(4,4'-dimethoxytrityl)-deoxythymidine-3'-[O-(2-cyanoethyl)]thiophosphoryldeoxythymidine-3'-yl[3,4,5-tri(octadecyloxy)benzyl]succinate (4.150 g, yield 99.8%) as a white solid.

[0785] Example 18: Continuous synthesis of phosphorothioate 20-mers (sequence: 5'-TCCCGCCTGTGACATGCATT-3')

[0786] (1) Synthesis of phosphorothioate 20-mers bonded with pseudo solid phase protecting groups

[0787] The operation similar to that of Example 17 was repeated 19 times to obtain 5'-O-(4,4'-dimethoxytrityl)-deoxythymidine 3'-[O-(2-cyanoethyl)]thiophosphoryl-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4-Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 2 -isobutyryl-deoxyguanosine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-deoxythymidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 2 -isobutyryl-deoxyguanosine 3'-[O-(2-cyanoethyl)]phosphorothioate-deoxythymidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 2 -isobutyryl-deoxyguanosine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 6 -Benzoyl-deoxyadenosine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 6 -Benzoyl-deoxyadenosine 3'-[O-(2-cyanoethyl)]phosphorothioate-deoxythymidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 2 -isobutyryl-deoxyguanosine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 4 -Benzoyl-deoxycytidine 3'-[O-(2-cyanoethyl)]phosphorothioate-N 6 -benzoyl-deoxyadenosine 3'-[O-(2-cyanoethyl)]phosphorothioyl-deoxythymidine 3'-[O-(2-cyanoethyl)]phosphorothioyl-deoxythymidine 3'-yl 3,4,5-tri(octadecyloxy)benzyl succinate (8.55 g).

[0788] (2) Synthesis (deprotection) and purification of phosphorothioate 20-mers

[0789] The compound synthesized in the above (1) (100 mg, 10.3 μmol) and 30 wt% ammonia water (5.00 mL) were placed in an autoclave and heated at 65°C for 4 hours. The insoluble matter was filtered through a syringe filter (WHATMAN 25 mm GD / X PTFE 0.45 μm). The filtrate was further diluted with 0.1 M aqueous ammonium acetate solution (50 mL) and purified by a C-18 column. The resulting eluate was freeze-dried to obtain the target product: deoxythymidine-[3'→5']-deoxycytidine-[3'→5']-deoxycytidine-[3'→5']-deoxycytidine-[3'→5']-deoxyguanidyl-[3'→5']-deoxycytidine-[3'→5']-deoxycytidine-[3'→5']-deoxyguanidyl Oxythymidine-[3'→5']-deoxyguanidino-[3'→5']-deoxythymidine-[3'→5']-deoxyguanidino-[3'→5']-deoxyadenyl-[3'→5']-deoxycytidyl-[3'→5']-deoxyadenyl-[3'→5']-deoxythymidine-[3'→5']-deoxyguanidino-[3'→5']-deoxycytidyl-[3'→5']-deoxythymidine-[3'→5']-deoxythymidine.

[0790] HPLC (shodexODP Flow rate 0.5 mL / min, 15 mM TEA + 20 mM HFIP / MeOH = 95 / 5, 15 mM TEA + 20 mM HFIP / MeOH = 50 / 50 Gradient: 0-7.4 min; 0-60%, 7.5-11 min; 100%, λ = 254 nm): Rt = 6.26 min (73%);

[0791] TOF / MS:6344.59[MH] -

[0792] Example 19: Continuous Synthesis of Phosphorothioate Dimers in Solution by Using a Precipitation Accelerator

[0793]

[0794] Under argon atmosphere, 5'-O-(4,4'-dimethoxytrityl)deoxythymidine-3'-yl-diisopropyl-4-[3,4,5-tri(octadecyloxy)benzyloxycarbonyl]phenylsilane (330 mg, 200 μmol) and 3,4,5-tri(octadecyloxy)benzyl acetate (290 mg, 303 μmol, which is the precipitation promoter synthesized in Example 7) were dissolved in dichloromethane (5.0 mL), indole (469 mg, 4.00 mmol) and trifluoroacetic acid (30.6 μL, 400 μmol) were added and the mixture was stirred at room temperature for 2 hr, and the completion of the reaction was confirmed by thin layer chromatography. After neutralizing the reaction mixture with 1-methylimidazole (34.8 μL, 439 μmol), 5-benzylthio-1H-tetrazole (115 mg, 600 μmol), 5'-O-(4,4'-dimethoxytrityl)deoxythymidine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (447 mg, 600 μmol) in acetonitrile were added, and the mixture was stirred at room temperature for 60 minutes, and the completion of the reaction was confirmed by thin layer chromatography. In addition, 3-[(N,N-dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione (136 mg, 662 μmol) was added and the mixture was stirred at room temperature for 30 minutes. Then acetonitrile (30 mL) was added to the reaction solution, and the precipitated solid was filtered using a Kiriyama funnel and then dried to obtain 5'-O-(4,4'-dimethoxytrityl)-deoxythymidine-3'-[O-(2-cyanoethyl)]thiophosphoryldeoxythymidine-3'-yl[3,4,5-tri(octadecyloxy)benzyl]succinate (398 mg, yield 98%) as a white solid.

[0795] 1 H-NMR (400MHz, CDCl3): δ=0.88(t,9H,J=6.6Hz,-CH3),1.00-1.07(m,12H,-Si-CH-(C H3)2 ),1.21-2.00(m,96H,-OCH2(C H2) 15 CH3),2.35-2.50(m,3H,2'-H),2.56-2.60(m,2H,2'-H+5'-H),2.71(m,1H,5'-H),3.41-3.46(m,2H,5'-H),3.78(s,6H,DMTr-OC H3 ),3.93-3.99(m,6H,-OC H2 (CH2) 15 CH3),4.11-4.30(m,6H,3'-H,4'-H,-C H2 C H2CN),4.51-4.60(m,1H,4'-H),5.24(s,2H,Ar-C H2 -O-),5.32(m,1H,3'-H),6.28(m,1H,1H'-H),6.36(m,1H,1H'-H),6.64(s,2H,Ar-H),6.84(m,4 H,DMTr-H),7.22-7.63(m,11H,-Si-Ar,DMTr-H),8.05-8.08(m,2H,-Si-Ar),8.58-8.95(m,2H,N 3 -H)

[0796] Example 20: Continuous Synthesis of Phosphorothioate Dimers in Solution by Using a Precipitation Accelerator

[0797]

[0798] Under argon atmosphere, 5'-O-(4,4'-dimethoxytrityl)-N3-3,4,5-tri(octadecyloxy)benzoyl-deoxythymidine-3'-yl-tert-butyl-dimethylsilane (312 mg, 199 μmol) and 3,4,5-tri(octadecyloxy)benzyl acetate (287 mg, 300 μmol, which is the precipitation promoter synthesized in Example 7) were dissolved in dichloromethane (5.0 mL), indole (466 mg, 3.98 mmol) and trifluoroacetic acid (30.6 μL, 398 μmol) were added and the mixture was stirred at room temperature for 2 hr, and the completion of the reaction was confirmed by thin layer chromatography. After neutralizing the reaction mixture with 1-methylimidazole (34.8 μL, 439 μmol), a solution of 5-benzylthio-1H-tetrazole (115 mg, 600 μmol) and 5'-O-(4,4'-dimethoxytrityl)deoxythymidine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (447 g, 600 μmol) in acetonitrile was added, and the mixture was stirred at room temperature for 60 min, and completion of the reaction was confirmed by thin layer chromatography. In addition, 3-[(N,N-dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione (136 mg, 662 μmol) was added and the mixture was stirred at room temperature for 30 min. Then acetonitrile (30 mL) was added to the reaction solution, and the precipitated solid was filtered using a Kiriyama funnel and then dried to obtain 5'-O-(4,4'-dimethoxytrityl)-deoxythymidine-3'-[O-(2-cyanoethyl)]thiophosphoryldeoxythymidine-3'-yl[3,4,5-tri(octadecyloxy)benzyl]succinate (382 mg, yield 99%) as a white solid.

[0799] 1H-NMR (400MHz, CDCl3): δ=-0.01(S,3H,Si-CH3),0.00(S,3H,Si-CH3),0.75-0.81(m,18H,-OCH2(CH2) 15 C H3 +Si-C(C H3)3 ),1.05-1.73(m,90H,-OCH2(C H2) 15 CH3),2.08(m,1H,2'-H),2.22(m,1H,2'-H),2.34(m,1H,2'-H),2.53-2.62(m,2H,2'-H+5'-H),2.70(t ,1H,J=6.0Hz,5'-H),3.25-3.51(m,2H,5'-H),3.71(d,6H,J=1.3Hz,DMTr-OCH3),3.87-3.98(m,7H,-OC H2 (CH2) 15 CH3+3'-H),4.10-4.33(m,6H,-C H2 C H2 CN+2×4'-H),5.25(dt,1H,J=7.9,15.3Hz,3'-H),6.17(td,1H,J=2.2,6.6Hz,1'-H),6.30(dd,J =5.4,8.8,23.6Hz,1'-H),6.76(s,2H,Ar-H),6.78(s,2H,DMTr-H),7.05-7.39(m,11H,DMTr-Ar- H ),7.39(s,1H,N6-H),7.47(d,1H,J=8.0Hz,N6-H),8.45(m,1H,N3-H)

[0800] [Industrial Applicability]

[0801] The precipitation accelerator of the present invention can improve the recovery rate of an organic compound protected by an organic group having one or more aliphatic hydrocarbon groups having 10 or more carbon atoms when the organic compound is precipitated from a solvent. Therefore, the precipitation accelerator of the present invention can be used for the synthesis of the organic compound, especially the synthesis of oligonucleotides.

[0802] This application is based on patent application No. 2015-009720 filed in Japan, the entire contents of which are incorporated herein by reference. Sequence Listing <110> Ajinomoto Co., Ltd. <120> Precipitation accelerator and precipitation method using the same <130> 092395 <150> JP 2015-009720 <151> 2015-01-21 <160> 1 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 1 tcccgcctgt gacatgcatt 20

Claims

1. A method for precipitating an organic compound protected by an organic group from a solvent, the method using a precipitation accelerator, wherein: The solvent comprises a polar solvent, The precipitation accelerator has one or more straight-chain aliphatic hydrocarbon groups having 10 or more carbon atoms, and the total number of carbon atoms of the aliphatic hydrocarbon groups of the precipitation accelerator is 20 or more, The precipitation promoter is a compound represented by formula (I), In the formula, R 1 Each independently represents a straight chain C 10-30 alkyl; X 1 each independently represents a single bond, -O-, -C(=O)-, -C(=O)O-, or -OC(=O)-; Ring A 1 and Ring B 1 Each independently represents a benzene ring or a cyclohexane ring; X 2 Each independently represents -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -, or -(CH2) p -OC(=O)-(CH2) q -, wherein p and q each independently represent an integer from 0 to 3; X 3 Represents a single bond, -(CH2) r -O-, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O-, or -(CH2) r -OC(=O)-, wherein r represents an integer from 0 to 3; R 2 represents a hydrogen atom, optionally replaced by C 1-6 Alkanoylamino substituted C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-14 aryl, 3-8 membered monocyclic non-aromatic heterocyclic group or tri(C 1-6 Alkyl)silyl; n represents 1 or 2; m represents an integer from 1 to 3; and When m is 1, m' represents 0 or 1; and when m is 2 or 3, m' represents 1, The organic group is a group represented by formula (III), ** LYZ (III) In the formula, ** Indicates the bonding position to the protected group; L represents a single bond, or a group represented by formula (a1) or (a1'): In the formula, * represents the bonding position with Y; ** Same as the above definition; R 8 and R 9 Each independently represents C 1-22 Hydrocarbon; L1 indicates divalent C 1-22 a hydrocarbon group; and L2 represents a single bond, or ** C(=O)N(R 2’ )-R 1’ -N(R 3 ) *** The group shown in the formula ** represents the bonding position with L1, *** Indicates the bonding position with C=O, R 1’ Represents C 1-22 Alkylene, R 2’ and R 3 Each independently represents a hydrogen atom or a C 1-22 alkyl; Y represents a single bond, an oxygen atom or NR, wherein R represents a hydrogen atom, an alkyl group or C 7-16 Aralkyl; and Z represents a group represented by formula (a2), formula (a2') or formula (a2"): In the formula, * Indicates the bonding position; R 4 is a hydrogen atom, or when R b When it is a group represented by the following formula (a3), R 4 Optionally with R of ring C 6 Together they represent a single bond or -O-, and together with Ring A or Ring B and Ring C form a condensed ring, wherein the condensed ring is a fluorene ring or a xanthene ring; k Qs each independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-; k R 5 Each independently represents a straight chain C 10-40 alkyl; k represents an integer from 1 to 4; Ring A and Ring B each independently have k QR 5 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C 1-6 Alkoxy; R a represents a hydrogen atom, or a phenyl group which is optionally substituted by a halogen atom; and R b represents a hydrogen atom, or a group represented by formula (a3): In the formula, * Indicates the bonding position; j represents an integer from 0 to 4; j Qs each independently represent the same meaning as above; j R 7 Each independently represents a hydrocarbon group to which an aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group; R 6 represents a hydrogen atom, or optionally connected to R of ring A or ring B 4 together represent a single bond or -O-, and together with ring A or ring B and ring C form a condensed ring; and In addition to having j QR 7 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C 1-6 Alkoxy, or R a and R b Together they form a carbonyl group, The organic compound is a nucleoside, nucleotide or oligonucleotide optionally further protected by a protecting group used in nucleic acid synthesis, or an amino acid or peptide optionally further protected by a protecting group used in peptide synthesis.

2. The method according to claim 1, wherein: R 2 is a hydrogen atom, optionally replaced by C 1-6 Alkanoylamino substituted C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-14 Aryl or 3-8 membered monocyclic non-aromatic heterocyclic group.

3. The method according to claim 1, wherein: Z is a group represented by formula (a2) or formula (a2'), k R 5 Each independently is a straight chain C 10-40 Alkyl, j R 7 are each independently a hydrocarbon group to which a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group, and R a is a hydrogen atom, or R a and R b together to form a carbonyl group.

4. The method according to claim 1, wherein: L is a group represented by formula (a1), L1 represents a divalent C 1-22 A hydrocarbon group, and L2 represents a single bond.

5. The method according to claim 1, wherein: L is a group represented by formula (a1′), L1 represents a phenylene group, and L2 represents a single bond.

6. The method according to claim 1, wherein: Y is an oxygen atom.

7. The method according to claim 1, wherein: Z is a group represented by formula (a2), R 4 A hydrogen atom.

8. The method according to claim 1, wherein: Z is a group represented by formula (a2), R a and R b Each is a hydrogen atom.

9. The method according to claim 1, wherein: L and Y are each a single bond, Z is a group represented by formula (a2), R 4 is a hydrogen atom, and R a and R b together to form a carbonyl group.

10. The method according to claim 1, wherein: The organic compound is a nucleoside, nucleotide or oligonucleotide which is optionally further protected by a protecting group used in nucleic acid synthesis.

11. The method according to claim 1, wherein: The organic compound protected by an organic group is a nucleoside or an oligonucleotide, in which at least one group selected from the amino and imino groups of nucleic acid bases, the 2' and 3' hydroxyl groups of ribose residues, and the 3' hydroxyl group of deoxyribose residues is protected by an organic group, and other groups are optionally further protected by protecting groups used in nucleic acid synthesis.

12. The method according to claim 1, wherein: The solvent containing a polar solvent is a mixed solvent of a polar solvent and a non-polar solvent.

13. The method according to claim 1, wherein: The polar solvent is acetonitrile.

14. The method according to claim 1, wherein: The organic compound protected by an organic group is precipitated by using the precipitation accelerator in an amount of 0.1 molar equivalent or more relative to the organic compound protected by an organic group.

15. A precipitation mixture comprising a precipitation accelerator and an organic compound protected by an organic group, wherein: The precipitation accelerator has one or more straight-chain aliphatic hydrocarbon groups having 10 or more carbon atoms, and the total number of carbon atoms of the aliphatic hydrocarbon groups of the precipitation accelerator is 20 or more, The precipitation promoter is a compound represented by formula (I), In the formula, R 1 Each independently represents a straight chain C 10-30 alkyl; X 1 each independently represents a single bond, -O-, -C(=O)-, -C(=O)O-, or -OC(=O)-; Ring A 1 and Ring B 1 Each independently represents a benzene ring or a cyclohexane ring; X 2 Each independently represents -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -, or -(CH2) p -OC(=O)-(CH2) q -, wherein p and q each independently represent an integer from 0 to 3; X 3 Represents a single bond, -(CH2) r -O-, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O-, or -(CH2) r -OC(=O)-, wherein r represents an integer from 0 to 3; R 2 represents a hydrogen atom, optionally replaced by C 1-6 Alkanoylamino substituted C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-14 aryl, 3-8 membered monocyclic non-aromatic heterocyclic group or tri(C 1-6 Alkyl)silyl; n represents 1 or 2; m represents an integer from 1 to 3; and When m is 1, m' represents 0 or 1; and when m is 2 or 3, m' represents 1, The organic group is a group represented by formula (III), ** LYZ (III) In the formula, ** Indicates the bonding position to the protected group; L represents a single bond, or a group represented by formula (a1) or (a1'): In the formula, * represents the bonding position with Y; ** Same as the above definition; R 8 and R 9 Each independently represents C 1-22 Hydrocarbon; L1 indicates divalent C 1-22 a hydrocarbon group; and L2 represents a single bond, or ** C(=O)N(R 2’ )-R 1’ -N(R 3 ) *** The group shown in the formula ** represents the bonding position with L1, *** Indicates the bonding position with C=O, R 1’ Represents C 1-22 Alkylene, R 2’ and R 3 Each independently represents a hydrogen atom or a C 1-22 alkyl; Y represents a single bond, an oxygen atom or NR, wherein R represents a hydrogen atom, an alkyl group or C 7-16 Aralkyl; and Z represents a group represented by formula (a2), formula (a2') or formula (a2"): In the formula, * Indicates the bonding position; R 4 is a hydrogen atom, or when R b When it is a group represented by the following formula (a3), R 4 Optionally with R of ring C 6 Together they represent a single bond or -O-, and together with Ring A or Ring B and Ring C form a condensed ring, wherein the condensed ring is a fluorene ring or a xanthene ring; k Qs each independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-; k R 5 Each independently represents a straight chain C 10-40 alkyl; k represents an integer from 1 to 4; Ring A and Ring B each independently have k QR 5 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C 1-6 Alkoxy; R a represents a hydrogen atom, or a phenyl group which is optionally substituted by a halogen atom; and R b represents a hydrogen atom, or a group represented by formula (a3): In the formula, * Indicates the bonding position; j represents an integer from 0 to 4; j Qs each independently represent the same meaning as above; j R 7 Each independently represents a hydrocarbon group to which an aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group; R 6 represents a hydrogen atom, or optionally connected to R of ring A or ring B 4 together represent a single bond or -O-, and together with ring A or ring B and ring C form a condensed ring; and In addition to having j QR 7 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C 1-6 Alkoxy, or R a and R b Together they form a carbonyl group, The organic compound is a nucleoside, nucleotide or oligonucleotide optionally further protected by a protecting group used in nucleic acid synthesis, or an amino acid or peptide optionally further protected by a protecting group used in peptide synthesis.

16. A method for preparing an oligonucleotide, the method comprising: a step of adding a polar solvent to a reaction solution containing an oligonucleotide in which at least one group is protected by an organic group and other groups are optionally further protected by protecting groups used in nucleic acid synthesis and a precipitation accelerator in a non-polar solvent, and separating a precipitation mixture containing the oligonucleotide and the precipitation accelerator from the reaction solution, wherein the precipitation accelerator has one or more straight-chain aliphatic hydrocarbon groups having 10 or more carbon atoms, and the total number of carbon atoms of the aliphatic hydrocarbon groups of the precipitation accelerator is 20 or more, The precipitation promoter is a compound represented by formula (I), In the formula, R 1 Each independently represents a straight chain C 10-30 alkyl; X 1 each independently represents a single bond, -O-, -C(=O)-, -C(=O)O-, or -OC(=O)-; Ring A 1 and Ring B 1 Each independently represents a benzene ring or a cyclohexane ring; X 2 Each independently represents -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -, or -(CH2) p -OC(=O)-(CH2) q -, wherein p and q each independently represent an integer from 0 to 3; X 3 Represents a single bond, -(CH2) r -O-, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O-, or -(CH2) r -OC(=O)-, wherein r represents an integer from 0 to 3; R 2 represents a hydrogen atom, optionally replaced by C 1-6 Alkanoylamino substituted C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-14 aryl, 3-8 membered monocyclic non-aromatic heterocyclic group or tri(C 1-6 Alkyl)silyl; n represents 1 or 2; m represents an integer from 1 to 3; and When m is 1, m' represents 0 or 1; and when m is 2 or 3, m' represents 1, Furthermore, the organic group is a group represented by formula (III), ** LYZ (III) In the formula, ** Indicates the bonding position to the protected group; L represents a single bond, or a group represented by formula (a1) or (a1'): In the formula, * represents the bonding position with Y; ** Same as the above definition; R 8 and R 9 Each independently represents C 1-22 Hydrocarbon; L1 indicates divalent C 1-22 a hydrocarbon group; and L2 represents a single bond, or represents **C(=O)N(R 2’ )-R 1’ -N(R 3 ) *** The group shown in the formula ** represents the bonding position with L1, *** Indicates the bonding position with C=O, R 1’ Represents C 1-22 Alkylene, R 2’ and R 3 Each independently represents a hydrogen atom or a C 1-22 alkyl; Y represents a single bond, an oxygen atom or NR, wherein R represents a hydrogen atom, an alkyl group or C 7-16 Aralkyl; and Z represents a group represented by formula (a2), formula (a2') or formula (a2"): In the formula, * Indicates the bonding position; R 4 is a hydrogen atom, or when R b When it is a group represented by the following formula (a3), R 4 Optionally with R of ring C 6 Together they represent a single bond or -O-, and together with Ring A or Ring B and Ring C form a condensed ring, wherein the condensed ring is a fluorene ring or a xanthene ring; k Qs each independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-; k R 5 Each independently represents a straight chain C 10-40 alkyl; k represents an integer from 1 to 4; Ring A and Ring B each independently have k QR 5 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C 1-6 Alkoxy; R a represents a hydrogen atom, or a phenyl group which is optionally substituted by a halogen atom; and R b represents a hydrogen atom, or a group represented by formula (a3): In the formula, * Indicates the bonding position; j represents an integer from 0 to 4; j Qs each independently represent the same meaning as above; j R 7 Each independently represents a hydrocarbon group to which an aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group; R 6 represents a hydrogen atom, or optionally connected to R of ring A or ring B 4 together represent a single bond or -O-, and together with ring A or ring B and ring C form a condensed ring; and In addition to having j QR 7 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C 1-6 Alkoxy, or R a and R b together to form a carbonyl group.

17. The preparation method according to claim 16, wherein: The polar solvent is acetonitrile.

18. The preparation method according to claim 16, which is carried out by a phosphoramidite method.

19. A method for preparing an oligonucleotide, which is a method for preparing an oligonucleotide using a phosphoramidite method, wherein the phosphoramidite method comprises performing a preparation cycle comprising the following steps (1) to (3) once or repeatedly for a plurality of times, in, The first cycle includes the following step (4), each cycle includes the following step (5), and each cycle except the final cycle includes the following step (6), (1) adding an acid to a reaction solution containing a nucleoside or an oligonucleotide in a non-polar solvent to deprotect the temporary protecting group of the 5'-hydroxyl group, and neutralizing the solution with a base to obtain a reaction solution containing a free form of the 5'-hydroxyl group, In the nucleoside or oligonucleotide, at least one group selected from the group consisting of the amino and imino groups of the nucleic acid bases, the 2' and 3' hydroxyl groups of the ribose residues, and the 3' hydroxyl group of the deoxyribose residues is protected by an organic group, the 5' hydroxyl group is protected by a temporary protecting group that is removable under acidic conditions, and the other groups are optionally further protected by protecting groups used in nucleic acid synthesis; (2) a step of adding a nucleoside or an oligonucleotide to a reaction solution containing a free 5'-hydroxyl group in a non-polar solvent to obtain a reaction solution containing a triester phosphite compound, In the nucleoside or oligonucleotide, the 3'-hydroxyl group is phosphoramidated, and the 5'-hydroxyl group is protected by a temporary protecting group that can be removed under acidic conditions, and other groups are optionally further protected by protecting groups used in nucleic acid synthesis; (3) adding an oxidizing agent or a sulfurizing agent to a reaction solution containing a triester phosphite compound in a non-polar solvent to obtain a reaction solution containing an oligonucleotide, In the oligonucleotide, at least one group selected from the group consisting of the amino and imino groups of the nucleic acid bases, the 2' and 3' hydroxyl groups of the ribose residues, and the 3' hydroxyl group of the deoxyribose residues is protected by the organic group, and the 5' hydroxyl group is protected by the temporary protecting group removable under acidic conditions, and other groups are optionally further protected by the protecting groups used in the nucleic acid synthesis; (4) a step of adding a precipitation accelerator to the reaction solution at any time before step (1), between steps (1) and (2), between steps (2) and (3), and after step (3); (5) after step (4) and at any time between steps (1) and (2), between steps (2) and (3), and after step (3), a step of adding a polar solvent to the reaction solution containing the precipitation accelerator to separate a precipitation mixture containing the 5'-hydroxyl free body, the triester phosphite compound or the oligonucleotide, and the precipitation accelerator from the reaction solution; (6) adding a non-polar solvent to the precipitate mixture obtained in step (5) to prepare a reaction solution, wherein the precipitation accelerator has one or more straight-chain aliphatic hydrocarbon groups having 10 or more carbon atoms, and the total number of carbon atoms of the aliphatic hydrocarbon groups of the precipitation accelerator is 20 or more, The precipitation promoter is a compound represented by formula (I), In the formula, R 1 Each independently represents a straight chain C 10-30 alkyl; X 1 each independently represents a single bond, -O-, -C(=O)-, -C(=O)O-, or -OC(=O)-; Ring A 1 and Ring B 1 Each independently represents a benzene ring or a cyclohexane ring; X 2 Each independently represents -(CH2) p -、-(CH2) p -O-(CH2) q -、-(CH2) p -C(=O)-(CH2) q -、-(CH2) p -C(=O)O-(CH2) q -, or -(CH2) p -OC(=O)-(CH2) q -, wherein p and q each independently represent an integer from 0 to 3; X 3 Represents a single bond, -(CH2) r -O-, -(CH2) r -C(=O)-, -(CH2) r -C(=O)O-, or -(CH2) r -OC(=O)-, wherein r represents an integer from 0 to 3; R 2 represents a hydrogen atom, optionally replaced by C 1-6 Alkanoylamino substituted C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-14 aryl, 3-8 membered monocyclic non-aromatic heterocyclic group or tri(C 1-6 Alkyl)silyl; n represents 1 or 2; m represents an integer from 1 to 3; and When m is 1, m' represents 0 or 1; and when m is 2 or 3, m' represents 1, Furthermore, the organic group is a group represented by formula (III), ** LYZ (III) In the formula, ** Indicates the bonding position to the protected group; L represents a single bond, or a group represented by formula (a1) or (a1'): In the formula, * represents the bonding position with Y; ** Same as the above definition; R 8 and R 9 Each independently represents C 1-22 Hydrocarbon; L1 indicates divalent C 1-22 a hydrocarbon group; and L2 represents a single bond, or ** C(=O)N(R 2’ )-R 1’ -N(R 3 ) *** The group shown in the formula ** represents the bonding position with L1, *** Indicates the bonding position with C=O, R 1’ Represents C 1-22 Alkylene, R 2’ and R 3 Each independently represents a hydrogen atom or a C 1-22 alkyl; Y represents a single bond, an oxygen atom or NR, wherein R represents a hydrogen atom, an alkyl group or C 7-16 Aralkyl; and Z represents a group represented by formula (a2), formula (a2') or formula (a2"): In the formula, * Indicates the bonding position; R 4 is a hydrogen atom, or when R b When it is a group represented by the following formula (a3), R 4 Optionally with R of ring C 6 Together they represent a single bond or -O-, and together with Ring A or Ring B and Ring C form a condensed ring, wherein the condensed ring is a fluorene ring or a xanthene ring; k Qs each independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-; k R 5 Each independently represents a straight chain C 10-40 alkyl; k represents an integer from 1 to 4; Ring A and Ring B each independently have k QR 5 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C 1-6 Alkoxy; R a represents a hydrogen atom, or a phenyl group which is optionally substituted by a halogen atom; and R b represents a hydrogen atom, or a group represented by formula (a3): In the formula, * indicates the bonding position; j represents an integer from 0 to 4; j Qs each independently represent the same meaning as above; j R 7 Each independently represents a hydrocarbon group to which an aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linking group; R 6 represents a hydrogen atom, or optionally connected to R of ring A or ring B 4 together represent a single bond or -O-, and together with ring A or ring B and ring C form a condensed ring; and In addition to having j QR 7 In addition, it may further have a substituent selected from a halogen atom, a C 1-6 alkyl, and C 1-6 Alkoxy, or R a and R b together to form a carbonyl group.

20. The preparation method according to claim 19, wherein: The polar solvent is acetonitrile.

21. The preparation method according to claim 19, wherein Steps (5) and (6) are performed after step (3).

22. The preparation method according to claim 19, further comprising the following step (7): (7) A step of removing all protecting groups of the oligonucleotide and isolating the oligonucleotide.

23. The preparation method according to claim 19, wherein: The non-polar solvent is a solvent selected from the group consisting of halogen solvents, aromatic solvents, ester solvents, aliphatic solvents, and combinations thereof.

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