Selectively soluble tricarboxylate acid-derived supports for liquid-phase oligonucleotide synthesis

Abstract

The present invention provides compounds useful as selectively soluble supports (or tags) for oligonucleotide synthesis that can be removed in a high yield under basic conditions and can afford a resulting product at a high purity in an organic synthesis reaction such as peptide synthesis, oligonucleotide synthesis, and the like. The protecting reagent is a selectively soluble compound of Formula (I), which is derived from a tricarboxylic acid.

Description

(P101781WO01) SELECTIVELY SOLUBLE TRICARBOXYLATE ACID-DERIVED SUPPORTS FOR LIQUID-PHASE OLIGONUCLEOTIDE SYNTHESIS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 690,605 filed on September 4, 2024, which is incorporated by reference herein in its entirety. BACKGROUND OF THE INVENTION

[0002] The present invention relates to selectively soluble supports for liquid-phaseoligonucleotide synthesis. The selectively soluble supports (also referred to herein as “tags”) are useful for precipitating an organic compound (e.g., a peptide, an oligonucleotide) in a solution wherein the organic compound uses the supports as a protecting group in oligonucleotide synthesis, particularly liquid phase synthesis, and methods of synthesis using such a compound.

[0003] Despite the widespread adoption of solid-phase synthesis of oligonucleotides dueto the ease of use and automation, the method encounters significant challenges when scaling up. These challenges include inefficient diffusion of reactants to the solid support, the necessity for large excesses of reagents to drive reactions to completion, and the physical limitations of solid supports that lead to heterogeneous reaction conditions. Moreover, the solid phase method often results in a loss of yield and purity with increased scale, necessitating complex and costly purification steps.

[0004] Conversely, liquid-phase synthesis on a soluble support offers a compellingalternative for scaling up oligonucleotide production. The soluble nature of the support in liquid-phase synthesis ensures homogenous reaction conditions, facilitating more efficient reactant interactions and reducing the requirement for excess reagents. This can lead to a more cost-effective process by minimizing waste and improving the overall yield and purity of the synthesized oligonucleotides.(P101781WO01)

[0005] Additionally, the liquid-phase approach can inherently accommodate largerreaction volumes without the physical constraints imposed by solid supports, thus enabling the synthesis of oligonucleotides at a larger scale. The ease of separating product from the soluble support through precipitation and washing steps circumvents the diffusion limitations of solid phase synthesis, offering a streamlined and potentially automatable purification process.

[0006] In an attempt to perform reactions in a homogeneous liquid phase while utilizingthe advantages of the solid phase method in that isolation and purification after the reaction can be performed by filtration and washing alone, a method of isolating a particular component dissolved in a liquid as a solid has been used. This is because precipitation of a particular component alone facilitates isolation and purification after reaction.

[0007] A particular component dissolved in a solution can be precipitated only whenpredetermined conditions, such as chemical properties, property and relationship with solvents of the compound, are satisfied. However, determination of precipitation conditions requires trial and error and experimental searching in most cases. In liquid phase synthesis, moreover, some compounds to be synthesized are insoluble in organic solvents used for extraction or show low solubility therein, which necessitates confirmation of the property of each compound to search for isolation and purification methods therefor. Particularly, when sequential and multistep synthesis reactions are required as in oligonucleotide synthesis and the like, since isolation and purification conditions such as precipitation, extraction and the like need to be determined based on the properties unique to the compound synthesized in each step, a long time and high cost are required.

[0008] To solve such problems, a method using a carrier molecule wherein a dissolvedstate and an insolubilized state (precipitated state) irreversibly change according to the varying solvent composition has been developed. Using such a carrier, an isolation target compound can be selectively precipitated from a homogeneous solution state, in other words, a particular compound can be isolated after a liquid phase reaction when other soluble components still remain in a solution, thus obviating the need to consider extraction and precipitation conditions for each compound.(P101781WO01)

[0009] Soluble supports for oligonucleotide synthesis have been engineered to enhancethe efficiency and scalability of the synthesis process. β-Cyclodextrin supports, for instance, offer a streamlined chromatography and extraction process for short 5-mer DNA oligonucleotides, but may be limited by oligomer length. Pentaerythritol-based supports boast versatility for both DNA and RNA syntheses up to 20 nucleotides, though the purification process is relatively complex. The adamantane-core support is notable for facilitating the synthesis of densely branched 8-mer oligonucleotides, yet the method may require extensive purification steps. PEG-based supports accommodate longer chains of up to 20-mers and are known for their efficient synthesis, though the need for multiple precipitation steps could be seen as a limitation in terms of process throughput and operational simplicity. ASS-based and Ajiphase supports have gained use in large scale production, providing a balance between efficiency and molecule stability.

[0010] Still there is a need in the art for new selectively soluble supports for liquid phaseorganic synthesis of oligonucleotides which are superior in broad utility and stability, that are useful as a protecting group (anchor) for oligonucleotide synthesis in the liquid phase. BRIEF SUMMARY OF THE INVENTION

[0011] In one aspect, disclosed herein is a selectively soluble support compoundrepresented by Formula (I): ,R3is H or C1-C40alkyl; and(P101781WO01) Y is selected from the group consisting of H, succinate, -C(O)-X, -C1-C10 alkyl-C(O)-X, - C(O)-(C1-C10alkyl)-C(O)-X, and -C(O)-aryl-C(O)-X, wherein X is Cl, Br, I, F, imidazole, or another leaving group.

[0012] Compounds of Formula (I) can be used as both a selectively soluble support foroligonucleotide synthesis and as a protecting agent for a functional group, such as a carboxy group, a hydroxy group, an amino group, and a mercapto group. The compound in which hydroxyl group in the case of oligonucleotides, is protected and supported by a compound of Formula (I) can have high solubility in a selected solvent. Accordingly, a compound in which a functional group is protected by a compound of Formula (I) and used as a protecting agent can be readily dissolved in an organic solvent and can be easily separated and purified by a procedure such as liquid-liquid phase separation. The protecting group used in the inventive compound can be readily eliminated by, for example, an acid, a base, or catalytic hydrogenation.

[0013] Compounds which can be protected by a compound of Formula (I) may be anycompound having a functional group such as a carboxy group, a hydroxy group, a diol group, and an amino group, and examples thereof include amino acids, peptides, saccharides, proteins, nucleotides, various medicinal compounds and agrochemical compounds, and various polymers and dendrimers.

[0014] This procedure using the selectively soluble support compounds of Formula (I)can also be used to synthesize an oligonucleotide in a liquid phase synthesis. Accordingly, in another aspect, disclosed herein is a liquid phase method of producing an oligonucleotide comprising a step of subjecting a 5′-hydroxyl group or 3′-hydroxyl group of a nucleoside or oligonucleotide having a selectively soluble support in at least one location selected from the group consisting of 2′-position, 3′-position, 5′-position and a nucleobase moiety and having a 5′-hydroxyl group or a 3′-hydroxyl group, to H- phosphonation to convert the 5′-hydroxyl group or the 3′-hydroxyl group into an H- phosphonated form, wherein the selectively soluble support is a compound represented by Formula (I):(P101781WO01) O R2, whereinR1is a straight or branched C6-C40aliphatic group; R2 is H or a straight or branched C1-C40 aliphatic group; R3is H or C1-C40alkyl; and Y is selected from the group consisting of H, succinate, -C(O)-X, -C1-C10 alkyl- C(O)-X, -C(O)-(C1-C10alkyl)-C(O)-X, and -C(O)-aryl-C(O)-X, wherein X is Cl, Br, I, F, imidazole, or another leaving group. Such method comprises at least one elongation reaction cycle comprising a step of subjecting a nucleoside or oligonucleotide having a selectively soluble support represented by Formula (I) in at least one location selected from the group consisting of 2′-position, 3′-position, 5′-position and a nucleobase moiety and having a 5′-hydroxyl group or a 3′-hydroxyl group, to H-phosphonation to convert the 5′- hydroxyl group or the 3′-hydroxyl group into an H-phosphonated form.

[0015] In certain aspects, the elongation reaction cycle comprises: a first step includingdeprotecting a first nucleoside or first oligonucleotide having a selectively soluble support represented by Formula (I) in at least one location selected from the group consisting of 2′- position, 3′-position and a nucleobase moiety, having a 3′-hydroxyl group protected with a basic protecting group or a selectively soluble support represented by Formula (I), and having a 5′-hydroxyl group protected with a temporary protecting group, to remove the temporary protecting group to form a 5′-hydroxyl group; a second step including converting the resultant 5′-hydroxyl group into an H-phosphonated form using an H- phosphonate reagent, and a third step of forming an oligomer of the first nucleoside or first oligonucleotide with a second nucleoside or second oligonucleotide having a 3′-hydroxyl group and having a 5′-hydroxyl group protected with a temporary protecting group, by forming a phosphite diester bond from the 5′-hydroxyl group, now converted to the H- phosphonated form, of the first nucleoside or first oligonucleotide and the 3′-hydroxyl group of the second nucleoside or second oligonucleotide.(P101781WO01)

[0016] The tags of Formula (I) allow for easy in-process NMR analysis for confirmation.

[0017] The embodiments and features of the present invention can be used alone or incombinations with each other. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The foregoing will be apparent from the following more particular description ofexample embodiments of the invention, as illustrated in the accompanying drawings.

[0019] FIG. 1 is an 1H NMR spectrum of compound 2 of the Examples in CDCl3;

[0020] FIG. 2 is an 1H NMR spectrum of compound 3 of the Examples in CDCl3;

[0021] FIG. 3 is an 1H NMR spectrum of compound 4 of the Examples in CDCl3;

[0022] FIG. 4 is an 1H NMR spectrum of compound 5 of the Examples in CDCl3;

[0023] FIG. 5 is an 1H NMR spectrum of compound 7 of the Examples (600 MHz,acetone-d6); and

[0024] FIG. 6 is an 1H NMR spectrum of compound 9 of the Examples (600 MHz,acetone-d6). DETAILED DESCRIPTION OF THE INVENTION

[0025] Unless otherwise specified in the sentences, any technique terms and scientificterms used in the present specification, have the same meaning as those generally understood by those of ordinary skill in the art in the art the present invention belongs to. Any methods and materials similar or equivalent to those described in the present specification can be used for practicing or testing the present invention, and preferable methods and materials are described in the following. All publications and patents referred to in the Specification are hereby incorporated by reference so as to describe and disclose constructed products and methodology described in, for example, publications usable in relation to the described invention.(P101781WO01) Definitions

[0026] As used herein, the terms "a", "an", "the" and the like refer to both the singularand plural unless the context clearly indicates otherwise. "A bottle", for example, refers to a single bottle or more than one bottle.

[0027] As used herein, the terms “comprising” (and any form of comprising, such as“comprise”, “comprises”, and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive and open-ended and include the options following the terms, and do not exclude additional, unrecited elements or method steps.

[0028] As used herein, the term “selectively soluble support” or “tag” is a compound that,when reacted with a nucleotide, protects a reactive site on the nucleotide and supports the elongation of the nucleotide and can be solubilized or precipitated by changing the composition of the solvent in which the elongation reactions occur.

[0029] As used herein, the term “alkyl” means a saturated hydrocarbon group which isstraight-chained or branched. In some embodiments, the alkyl group has from 1 to 20 carbon atoms, from 2 to 20 carbon atoms, from 1 to 10 carbon atoms, from 2 to 10 carbon atoms, from 1 to 8 carbon atoms, from 2 to 8 carbon atoms, from 1 to 6 carbon atoms, from 2 to 6 carbon atoms, from 1 to 4 carbon atoms, from 2 to 4 carbon atoms, from 1 to 3 carbon atoms, or 2 or 3 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, t- butyl, isobutyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), hexyl, isohexyl, heptyl, octyl, nonyl, 4,4-dimethylpentyl, 2,2,4-trimethylpentyl, decyl, undecyl, dodecyl, 2-methyl-1- propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2- methyl-1-pentyl, 2,2-dimethyl-1-propyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2- pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, and the like.

[0030] As used herein, the term “alkylamino” means an amino group substituted by atleast one alkyl group. In some embodiments, the alkyl group is a lower alkyl group having from 1 to 6 carbon atoms. Alkylamino groups include, but are not limited to, -NHCH2CH3, -NH(CH2)2CH3, -NH(CH2)3CH3, -NH(CH2)4CH3, and -NH(CH2)5CH3, and the like.(P101781WO01)

[0031] As used herein, the term “amino” means -NH2.

[0032] As used herein, the term “aryl” means a monocyclic, bicyclic, or polycyclic (e.g.,having 2, 3 or 4 fused rings) aromatic hydrocarbon. In some embodiments, the aryl group has from 6 to 20 carbon atoms or from 6 to 10 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and tetrahydronaphthyl, and the like.

[0033] As used herein, the term “aralkyl” or “arylalkyl” refers to a monovalent alkylgroup substituted with one or more aryl groups. In certain embodiments, the aralkyl has from 7 to 30, from 7 to 20, or from 7 to 16 carbon atoms. Examples of aralkyl groups include, but are not limited to, benzyl, 1-phenylethyl, 2-phenylethyl, and 3-phenylpropyl. In certain embodiments, the aralkyl is optionally substituted with one or more.

[0034] The term “halogen”, “halide” or “halo” refers to fluorine, chlorine, bromine,and / or iodine.

[0035] As used herein, the term “nucleoside” is the constituent unit of an oligonucleotidemeans a compound wherein a nucleic acid base is bonded to the 1′-position of a sugar (e.g., ribose or deoxyribose wherein the carbon atoms at 2-position and carbon atom at 4-position of 2-deoxyribose, ribose, ribose ring or deoxyribose ring are bonded by a divalent organic group, and the like) by N-glycosidation. Examples of the ribose or deoxyribose wherein the carbon atom at 2-position and carbon atom at 4-position of the ribose ring or deoxyribose ring are bonded by a divalent organic group include the following compounds:(P101781WO01) . wherein a phosphoric

[0037] As used herein, the term “oligonucleotide” means a compound wherein one ormore of a plurality of nucleotides are bonded to a nucleoside. While the number of nucleosides in oligonucleotide in the present invention is not particularly limited, it is preferably 3 to 50, more preferably 5 to 30. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and / or deoxyribonucleosides (DNA).

[0038] As used herein, the term “nucleoside” and “nucleotide” encompass morpholinonucleoside and morpholino nucleoside having a morpholine residue instead of a ribose residue or a deoxyribose residue.

[0039] The terms “linker” and “linkage” may be used interchangeably and mean achemical moiety comprising a covalent bond or a chain of atoms that covalently attaches, or is attached to, for example, a selectively soluble support disclosed herein to a nucleoside or an oligonucleotide.(P101781WO01)

[0040] Also as used herein, the description of one or more method steps does notpreclude the presence of additional method steps before or after the combined recited steps. Additional steps may also be intervening steps to those described. In addition, it is understood that the lettering of process steps or ingredients is a convenient means for identifying discrete activities or ingredients and the recited lettering can be arranged in any sequence.

[0041] Where a range of numbers is presented in the application, it is understood that therange includes all integers and fractions thereof between the stated range limits. A range of numbers expressly includes numbers less than the stated endpoints and those in-between the stated range. A range of from 1-3, for example, includes the integers one, two, and three as well as any fractions that reside between these integers. Compounds

[0042] The compounds of the present invention are selectively soluble supportcompounds represented by Formula (I): , wherein R1is aR2 is H or a straight or branched C1-C40 aliphatic group; R3is H or C1-C40alkyl; and Y is selected from the group consisting of H, succinate, -C(O)-X, -C1-C10 alkyl- C(O)-X, -C(O)-(C1-C10alkyl)-C(O)-X, and -C(O)-aryl-C(O)-X, wherein X is Cl, Br, I, F, imidazole, or another leaving group.

[0043] A compound represented by Formula (I) of the present invention is bonded to acompound to be supported and protected via group Y. That is, the compound of the present invention wherein Y is a H, succinate, -C(O)-X, -C1-C10 alkyl-C(O)-X, -C(O)-(C1-C10(P101781WO01) alkyl)-C(O)-X, and -C(O)-aryl-C(O)-X, is attached to the 3-OH terminal of a nucleoside, for example for support during elongation for the synthesis of the oligonucleotide.

[0044] The “aliphatic group” of R1 is an aliphatic hydrocarbon group consisting ofstraight chain or branched, saturated or unsaturated, and an aliphatic hydrocarbon group having a carbon number of not less than 6 is preferable, an aliphatic hydrocarbon group having a carbon number of 6 to 40 is particularly preferable, an aliphatic hydrocarbon group having a carbon number of 6 to 30 is more preferable, and an aliphatic hydrocarbon group having a carbon number of 10 to 30 is further preferable.

[0045] The “aliphatic group” of R2 is an aliphatic hydrocarbon group consisting ofstraight chain or branched, saturated or unsaturated, and an aliphatic hydrocarbon group having a carbon number of from 1 to 40 is preferable, an aliphatic hydrocarbon group having a carbon number of 1 to 20 is particularly preferable, an aliphatic hydrocarbon group having a carbon number of 1 to 10 is more preferable, and an aliphatic hydrocarbon group having a carbon number of 1 to 5 is further preferable. That being said, the R groups are sometimes referred to as “tails,” whose length controls the solubility of the compounds of Formula (I) and can be designed for different systems having different solvents or mixtures of solvents.

[0046] Examples of the “aliphatic group” include monovalent groups such as an alkylgroup, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group and the like and divalent groups derived therefrom. Preferred are monovalent groups such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, a hexyl group, an octyl group, a decyl group, a dodecyl group, a lauryl group, a tridecyl group, a myristyl group, a cetyl group, a stearyl group, an aralkyl group, a behenyl group, an oleyl group, an isostearyl group and the like and divalent groups derived therefrom. In preferred embodiments, R1is a stearyl group, C18H37.

[0047] Examples of the “alkyl group” for R3 include a C1-40 alkyl group, preferably a C1-10 alkyl group, more preferably a C1-6 alkyl group. Preferable examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, and the like, and particularly preferred are methyl and ethyl.(P101781WO01)

[0048] In some embodiments, Y is H and R1 is C18H37. In other embodiments, Y issuccinate and R1is C18H37.

[0049] In other embodiments, Y is H, R2 is a methyl group, and R1 is C18H37. In yet otherembodiments, R2 is a straight or branched C1-C40 aliphatic group, such as, for example, a straight C18H37chain.

[0050] In other embodiments, R1 is C18H37, and R3 is methyl. In yet other embodiments,Y is H, R2is a methyl group, and R3is H. In other embodiments, Y is selected from the group consisting of -COOH, -C(O)-X, and -O-C(O)-X; and X is imidazole.

[0051] Examples of a compound of Formula (I) include the compoundspresent invention can be madeby any method known to those skilled in the art. An exemplary method is detailed in O Me N C18H37O Me O NH .(P101781WO01)

[0053] Next, an oligonucleotide production method according to the present inventionwill be described. Specifically, there will be described a method in which a nucleoside or oligonucleotide (hereinafter, also written as the “n-mer oligonucleotide”) protected with a selectively soluble support of Formula (I) is elongated with a nucleoside or oligonucleotide (hereinafter, also written as the “p-mer oligonucleotide”) to produce an oligonucleotide (hereinafter, also written as the “(n+p)-mer oligonucleotide”) protected with a selectively soluble support of Formula (I). For oligonucleotide synthesis, the Y portion of the compounds of Formula (I) is preferably selected from the group consisting of H, succinate, -C(O)-X, -C1-C10 alkyl-C(O)-X, -C(O)-(C1-C10 alkyl)-C(O)-X, and -C(O)-aryl-C(O)-X, as defined above. This is to accommodate an ester linkage to the 3’ OH group on a nucleoside via an O-C(O)- linkage. The n-mer oligonucleotide means an oligonucleotide in which n nucleoside units are linked together via a phosphorus-containing group. When n is 1, the n-mer oligonucleotide is understood as a nucleoside. The same applies to the p- mer oligonucleotide.

[0054] The oligonucleotide production method includes a step of subjecting a nucleosideor oligonucleotide having a selectively soluble support of Formula (I) in at least one location selected from the group consisting of 2′-position, 3′-position, 5′-position and a nucleobase moiety and having a 5′-hydroxyl group or a 3′-hydroxyl group, to H- phosphonation to convert the 5′-hydroxyl group or the 3′-hydroxyl group into an H- phosphonated form.

[0055] Further, the oligonucleotide production method includes at least one elongationreaction cycle including the above H-phosphonation step.

[0056] The oligonucleotide production method is characterized in that the elongationreaction cycle includes the step of subjecting a nucleoside or oligonucleotide having a selectively soluble support of Formula (I) to H-phosphonation to introduce an H- phosphonated form in place of the 5′-hydroxyl group or the 3′-hydroxyl group. The oligonucleotide production method preferably includes the following Step a to Step d in the elongation reaction cycle. Step (a) to Step (d) are performed in the order of Step (a), Step (b), Step (c) and Step (d), or in the order of Step (a), Step (d), Step (b) and Step (c), and are preferably performed in the order of Step (a), Step (b), Step (c) and Step (d).(P101781WO01)

[0057] Step (a)

[0058] Step (a) is a temporary protecting group removal step including deprotecting anucleoside or oligonucleotide which has a selectively soluble support of Formula (I) on at least one of a hydroxyl group that is not the elongation terminal, a nucleobase moiety and 2′-position and which has at the elongation terminal a hydroxyl group protected with a temporary protecting group, to remove the temporary protecting group to form a hydroxyl group.

[0059] Step (b)

[0060] Step (b) is a phosphonation step including converting the hydroxyl group resultingfrom the removal of the temporary protecting group into an H-phosphonated form using an H-phosphonate reagent.

[0061] Step (c)

[0062] Step (c) is a coupling step including adding a nucleoside or oligonucleotide havinga hydroxyl group to the H-phosphonated nucleoside or oligonucleotide obtained in Step (b), and condensing the compounds by forming a phosphite diester bond via the hydroxyl group.

[0063] Step (d)

[0064] Step (d) is a conversion step including converting the phosphite diester bond intoa phosphodiester bond, a thiophosphodiester bond, an aminophosphodiester bond, a boranophosphodiester bond, a phosphodiester bond protected with a basic protecting group (a phosphotriester bond), a thiophosphodiester bond protected with a basic protecting group (a thiophosphate-O, O, S-triester bond) or the like.

[0065] Here, the amino group in the aminophosphodiester bond is unsubstituted or issubstituted with one or two C1-6alkyl groups.

[0066] The phosphodiester bond protected with a basic protecting group is a bond formedby substituting a phosphodiester bond with, for example, a C1-40 alkyl group, a C3-6 cycloalkyl group, a C6-10aryl group, a 5-10 membered heteroaryl group, an aralkyl group or a heteroaralkyl group in place of the hydrogen atom of the single hydroxyl group. Here, the C1-6alkyl group is unsubstituted, or is substituted with a substituent such as a halogen atom or a cyano group. The C3-6 cycloalkyl group, the C6-10 aryl group, the 5-10 membered(P101781WO01) heteroaryl group, the aralkyl group and the heteroaralkyl group are unsubstituted, or are substituted with a substituent such as a C1-6alkyl group, a halogen atom or a cyano group.

[0067] The thiophosphodiester bond protected with a basic protecting group is a bondformed by substituting a thiophosphodiester bond with, for example, a C1-40 alkyl group, a C3-6cycloalkyl group, a C6-10aryl group, a 5-10 membered heteroaryl group, an aralkyl group or a heteroaralkyl group in place of the hydrogen atom of the single thiol group. Here, the C1-40alkyl group is unsubstituted, or is substituted with a substituent such as a halogen atom or a cyano group. The C3-6 cycloalkyl group, the C6-10 aryl group, the 5-10 membered heteroaryl group, the aralkyl group and the heteroaralkyl group are unsubstituted, or are substituted with a substituent such as a C1-6 alkyl group, a halogen atom or a cyano group.

[0068] In the nucleoside or oligonucleotide with a selectively soluble support of Formula(I) that is used in Step (a), the number n of the nucleoside units is not particularly limited as long as it is an integer of 1 or greater, but is preferably 1 to 50, more preferably 1 to 30, still more preferably 1 to 20, even more preferably 1 to 10, and particularly preferably 1 to 5.

[0069] In the nucleoside or oligonucleotide with a hydroxyl group that is used in Step (c),the number p of the nucleoside units is not particularly limited as long as it is an integer of 1 or greater, but is preferably 1 to 50, more preferably 1 to 30, still more preferably 1 to 20, even more preferably 1 to 5, further preferably 1 to 3, and particularly preferably 1. That is, it is particularly preferable to use a nucleoside.

[0070] The reaction mixture obtained in Step (d) may be directly used in Step (a).Treatment such as heating may be performed appropriately after the completion of Step (d), and thereby Step (a) may be performed concurrently.

[0071] The oligonucleotide production method may further include the following Step(e), in which case the nucleoside or oligonucleotide may be purified simply and effectively by removing the excess of raw materials and byproducts.(P101781WO01)

[0072] Step (e)

[0073] Step (e) is a separation step of adding a polar solvent to the reaction mixtureobtained from any of Step (a) to Step (d) to precipitate the nucleoside or oligonucleotide having a pseudo solid phase-protecting group and collecting the precipitate by solid liquid separation.

[0074] Examples of the polar solvent include alcohol solvents such as methanol, ethanol,isopropanol and the like; nitrile solvents such as acetonitrile, propionitrile and the like; ketone solvents such as acetone, 2-butanone and the like; polar ether solvents such as 1,4- dioxane, tetrahydrofuran and the like; amide solvents such as dimethylformamide, dimethylacetamide, N-methylpiperidone and the like, sulfoxide solvents such as dimethyl sulfoxide and the like. Only one kind of polar solvent may be used, or two or more kinds thereof may be used in combination.

[0075] Step (e) is specific to the liquid phase synthesis method using a selectively solublesupport of Formula (I) and cannot be present in a usual liquid phase synthesis method using no selectively soluble support of Formula (I) or in a solid phase synthesis method.

[0076] The number of Steps (e) present in the elongation reaction cycle is not particularlylimited. Step (e) may be performed after any of Step (a) to Step (d).

[0077] For example, Step (e) may be performed 0 to 5 times, preferably 0 to 3 times,more preferably 0 to 2 times, and still more preferably 0 or 1 time independently after each of Step (a) to Step (d). Here, one or more Steps (e) are present after at least one of Steps (a) to (d).

[0078] The elongation reaction cycle preferably includes one to four Steps (e). Tomanage and control the generation of byproducts strictly and to lead the compounds to a high-purity oligonucleotide, it is preferable that Step (e) be performed one time after at least one of Step (a), Step (b) and Step (d) during the elongation reaction cycle. More preferably, Step (e) is performed one time after Step (b), one time after Step (d), or one time after each of Step (b) and Step (d) during the elongation reaction cycle.

[0079] In another embodiment, it is more preferable that Step (e) be performed one timeafter Step (a), one time after Step (b), or one time after each of Step (a) and Step (b), and it is still more preferable that Step (e) be performed one time after each of Step (a) and Step (b) during the elongation reaction cycle.(P101781WO01)

[0080] If the circumstances permit the amount of byproducts to be controlled bycontrolling the equivalents of the raw materials and by controlling the reaction, it is preferable that Step (e) be performed after Step (a) to Step (d) as a basic unit are repeated.

[0081] The oligonucleotide production method may further include Step (f), in whichcase the desired oligonucleotide can be isolated and produced.

[0082] Step (f)

[0083] Step (f) is a deprotection step of removing all of the base labile protecting groups,the temporary protecting group and the selectively soluble support of Formula (I) in the oligonucleotide obtained by Step (a) through Step (e).

[0084] In a final step (excluding any purification steps), the formed oligonucleotide iscleaved from the selectively soluble support of the present invention by addition of an acid or a base as is readily understood by a person of ordinary skill in the art. Typically, the oligonucleotide is removed from the support with concentrated ammonium hydroxide.

[0085] Specific examples of liquid phase oligonucleotide production with partiallysoluble supports can be found in, for example, U.S. Patent No.10,464,966, U.S. Publication No.2019 / 0169223, and U.S. Patent No.9,029,528, the entireties of which are incorporated herein by reference.

[0086] Composition Changing Means

[0087] As the means for changing the composition of a solution which is preferably usedin the method of the present invention, means capable of changing a composition of a solution dissolving a tag-protected oligonucleotide are used without restriction. The preferable means for changing a composition of a solution includes, for example, means in which a lower solubility solvent or antisolvent is added to a solution containing a dissolved tag-protected oligonucleotide as it is or the lower solubility solvent is added after concentrating the initial solvent of the solution, to cause precipitation. Here, concentrating means to distill off a part or all of the solvent. Thereafter, the precipitated tag-protected supported oligonucleotide can be separated, for example, by filtration and centrifugal separation. Impurities separated together with the precipitated tag-protected and supported oligonucleotide can be removed completely from the precipitated tag-protected supported oligonucleotide, preferably by washing the separated precipitate with an organic solvent(P101781WO01) which is selected to solubilize the impurities over the precipitated tag-protected supported oligonucleotide

[0088] The lower solubility antisolvent solvent used in the present invention means asolvent in which a tag-protected oligonucleotide or nucleotide is poorly soluble, that it, a tag-protected oligonucleotide is not dissolved easily or is not dissolved. For a tag-protected oligonucleotide that is not dissolved easily or is not dissolved, the poor solvent may advantageously be a solvent with which the solubility of a tag-protected oligonucleotide is less than 1% by mass at 25 °C, and preferable low solubility solvents are acetonitrile, any proportioned aqueous acetonitrile, methanol, or lower alkyl, alcohols and any proportioned aqueous methanol or lower alkyl alcohols (i.e., C1-C4) and water.

[0089] One skilled in the art, using the preceding detailed description, can utilize thepresent invention to its fullest extent. The following example are provided to illustrate the invention but should not be construed as limiting the invention in any way except as indicated in the appended claims. EXAMPLES Example 1: Synthesis of Exemplary Selectively Soluble Compound 5(P101781WO01) Nitro tri-amide (2) NO2O NMeC18H37O 21 eq) in DCM (36 mL, 12(50% wt / wt, 4 eq) was (3.1 eq) in was stirred at 25 ℃ with saturated NaHCO3 via silica gel g) as an off-white solid.(9H), 2.33 (12H), 1.53(6H), 1.30 (90 H), 0.91 (9H).

[0092] Amine (3)

[0093] To a solution of 2 in AcOH (7 vol) was charged Zn dust (12.5 eq). The mixturewas stirred at 25 ℃ for 20 h, filtered over celite, and washed with DCM (2 x 5 vol). The filtrate was concentrated to dryness followed by addition of heptane (8 vol) and concentration to dryness (repeated once). The residue was dissolved in DCM (10 vol), washed with saturated NaHCO3(5 vol), and concentrated to dryness to afford amine 3 in quantitative yield (5.71 g) as a light pink solid. FIG.2 shows the1H NMR of compound 3.

[0094] 1H NMR (CDCl3, 600 MHz) δ 3.35-3.25 (6H), 3.02-2.90 (9H), 2.37 (6H), 1.72(6H), 1.55 (6H), 1.30 (90 H), 0.91 (9H).

[0095] Succinate (4)(P101781WO01)

[0096] To a solution of amine 3 (5.87 g, 5.62 mmol, 1 eq) in DCM (35 mL, 6 vol) wascharged succinic anhydride (0.675 g, 6.75 mmol, 1.2 eq). After stirring the reaction mixture at 25 ℃ for 3 h, water (4 vol) was charged and stirring continued 1 h. The aqueous was removed and the DCM layer was concentrated to afford carboxylic acid 4 in 90% yield (5.80 g) as an off-white solid. FIG.3 shows the1H NMR of compound 4.

[0097] 1H NMR (CDCl3, 600 MHz) δ 3.38-3.25 (6H), 3.02-2.90 (9H), 2.68-2.50 (2H),2.41 (6H), 1.72 (6H), 2.1 and 1.7 (6H), 1.53 (6H), 1.30 (90 H), 0.91 (9H). a TFA(2.0 g) as an off-white solid. FIG.4 shows the1H NMR of compound 5.

[00100] 1H NMR (CDCl3, 600 MHz) δ 3.57 (2H), 3.47 (2H), 3.35-3.25 (6H), 3.05-2.85(9H), 2.90-2.80 (4H), 2.62 (2H), 2.46 (2H), 2.37 (6H), 2.10 (6H), 1.52 (6H), 1.28 (90H), 0.90 (9H). Example 2: Synthesis of a 7-Mer (Heptamer) OligonucleotideSoluble Support of Example 1(P101781WO01) O O O N NH O N NH O N O N N N DMTrO H N N O5 - Tag 8 Tag-3'-dG(iBu)-5'-OH NHBz NHBz N N O N O N O N NH ODMTrOOHOOO N N N H O N H O O NH O O NC O P NNCO O PSN N N H O O O N (DMT-dC(Bz) amidite) O O 1. DCI DCM, RT O2. Xanthane Hydride N 3. TFA, C H SH, 0±5 °CO NO HN O O O NMeC H NMeC H HN O O O C H MeN O NMeC H NMeC H 9 C H MeN O10Tag-3'-dG(iBu)-5'-OH Tag-3'-dG(iBu)-dC(Bz)-5'-OH(P101781WO01)(P101781WO01) Nucleoside Succinate (7) ,(P101781WO01) The mixture was stirred 5 hours. The DCM layer was washed three times with saturated 8 as d6) ,5.53, ,

[0107] To a stirred solution of 8 (0.8 g, 0.41 mmol, 1.0 eq) in DCM (4 mL) at 0±5 °C (icebath) was added 1-dodecanethiol (0.13 mL, 0.54 mmol, 1.3 eq). To the reaction trifluoroacetic acid (0.16 mL, 2.07 mol, 5.0 eq) was added dropwise. The reddish reaction mixture continued stirring at 0±5 °C. After 1 h, TLC showed the complete disappearance of 8. The reaction mixture was quenched by the reverse addition of reaction mixture to the stirred 10 wt% aq. K2CO3 (5 mL).2 mL DCM was used as a rinse. The organic layer was washed with brine (3 mL × 2). The organic layer was dried over anhy. magnesium sulphate, filtered. The filtrate was concentrated under vacuo to a thick gel.2 mL of DCM was added to dissolve and this solution was added to stirred MeCN (30 mL). The resulting slurry was stirred at RT for 1h. The slurry was filtered and the solid in the filter was dried under vacuum at RT for at least 1 h to obtain 9 as a white solid in 0.436 g (65% yield).(P101781WO01)

[0108] 1H-NMR (CDCl3, 600 MHz) δ 12.2 (br s, 1H), 8.0 (br s, 1H), 6.23 (s, 1H), 5.53(d, 1H), , 2.90 (s, , 0.89 (t,

[0109]

[0110] DCM(2.5 mL) this reaction mixture reactionthe complete conversion of 9. To the reaction was added xanthane hydride (0.043 g, 0.28 mmol, 1.1 eq). The reaction continued stirring at RT for 1 h. The IPC was analyzed by31P- NMR which showed the complete conversion. To the reaction was then added 1- dodecanethiol (0.21 mL, 0.85 mmol, 3.3 eq). The reaction was cooled to 0 ± 5 °C and TFA (0.20 mL, 2.57 mmol, 10 eq) was added dropwise. The resulting reddish reaction mixture continued stirring for 2 h. The reaction was quenched with the addition of NMI (0.245 mL, 0.29 mmol, 11 eq). The mixture was filtered, and the filtrate was further washed with 10 wt% aq. K2CO3(4 mL × 2) followed by brine (4 mL). The resulting organic layer was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was dissolved to a minimum of DCM (1 mL). The solution was added to the stirred MeCN (15 mL) at RT. The resulting slurry was stirred for 1 h, filtered and the solid was washed with MeCN (5 mL × 2). The solid was dried under vacuo at RT for 1 h. The light-yellow solid(P101781WO01) of 10 was obtained in 0.50 g (78%). The sample was analyzed by1HNMR which showed in . To underat RT for 1 h. DCI (0.039 g, 0.33 mmol, 1.5 eq) was added to the reaction and continued stirring for 3 h. To the reaction was added xanthane hydride (0.050 g, 0.33 mmol, 1.5 eq). The reaction continued stirring at RT for 3 h. The IPC was analyzed by31P-NMR which showed the complete conversion. The reaction mixture was filtered, and the filtrate was concentrated down to dryness. To this was added DCM (5 mL) and powdered activated 3 Å molecular sieve (0.42 g). The mixture was stirred for 45 minutes. To the reaction was then added 1-dodecanethiol (0.17 mL, 0.72 mmol, 3.3 eq). The reaction was cooled to 0 ± 5 °C and TFA (0.17 mL, 2.18 mmol, 10 eq) was added dropwise. The resulting reddish reaction mixture continued stirring for 3 h. The reaction was quenched with the addition of(P101781WO01) NMI (0.19 mL, 0.24 mmol, 11 eq). The mixture was filtered, and the filtrate was further The the the solid 0.5 h. with and 66.31. mixturemixture calcd for

[0115] To a stirred solution of DMT-dT amidite (0.13 g, 0.18 mmol, 1.5 eq) in DCM (2.5mL) was added 11 (0.34 g, 0.12 mmol, 1 eq) as a solution in DCM (2.5 mL). To the resulting solution was added further 5 mL of DCM was added and the solution was distilled under vacuum at 30 °C to azeotropically dry the solution. This azeotrope cycle(P101781WO01) was performed two additional times and concentrated down to 4 mL total solution. To this(P101781WO01)

[00118] To a stirred solution of DMT-dT amidite (0.11 g, 0.15 mmol, 1.5 eq) in DCM (2.5mL) was added 12 (0.30 g, 0.10 mmol, 1 eq) as a solution in DCM (2.5 mL). The solution was distilled under vacuum at 30 °C to azeotropically dry the solution. This azeotrope cycle was performed two additional times and concentrated down to 4 mL total solution. To this was added powdered activated 3 Å molecular sieve (0.32 g, 106 wt%). The mixture was stirred at RT. After 1 h, DCI (0.018 g, 0.15 mmol, 1.5 eq) was added to the reaction and continued stirring. The HPLC analysis showed the completion of reaction mmol, 1.1 eq). The NMR which To the filtrate was was cooled to 0 ± The resulting reddish was quenched layer was washed with MgSO4, filtered, of DCM (1 mL). Theslurry was stirred for(P101781WO01) 1 h, filtered and the solid was washed with MeCN (5 mL × 2). The solid was dried under .31P-NMR the mixturethe mixture calcd for

[0121] To a reaction vial was charged DMT-dC(Bz) amidite (0.08 g, 0.09 mmol, 1.5 eq),13 (0.20 g, 0.06 mmol, 1 eq), powdered activated 3 Å molecular sieve (0.25 g, 125 wt%) and DCM (3.0 mL). The resulting mixture was stirred at RT. After 1 h, DCI (0.011 g, 0.09 mmol, 1.5 eq) was added to the reaction and continued stirring at RT. After 3.5 h Xanthane hydride (0.010 g, 0.07 mmol, 1.1 eq) was added to the reaction. The reaction continued stirring at RT overnight. The IPC analyzed by 31P-NMR showed the complete conversion.(P101781WO01) To the reaction was then added 1-dodecanethiol (0.05 mL, 0.20 mmol, 3.3 eq). The h. The mL). dried to (2 mL 14 was 66.16,was stirred at 65 °C for 6 h. An aliquot was diluted with MeCN / H2O (1:1) and the mixture was filtered (0.2 µm filter). The filtrate was analyzed by LCMS. MS (ESI+): m / z calcd for 5’-HO-CTTGCG-OH-3’ [C58H75N20O31P5S5 + H]+: 1863.2, found: 1863.2.

[0123] Tag-2-3'-dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-dC(Bz)-dT-5'-OH (15)(P101781WO01) ,14at To 31P- TFA NMI 2). in. to the stirred MeCN (5.5 mL) at RT. The resulting slurry was stirred for 1 h, filtered and the(P101781WO01) solid was washed with MeCN (2 mL × 2). The solid was dried under vacuum at RT for 1 h. The light-yellow solid of 15 was isolated in 0.13 g (78%).31P-NMR (CDCl3, 242.9 MHz) 66.54, 66.33, 66.17.

[0125] 5 mg of the solid 15 was treated with NH4OH (0.2 mL, 28-30%) and the mixturewas stirred at 65 °C for 5 h. An aliquot was diluted with MeCN / H2O (1:1) and the mixture was filtered (0.2 µm filter). The filtrate was analyzed by LCMS. MS (ESI+): m+z / z calcd for 5’-HO-TCTTGCG-OH-3’ [C68H88N22O37P6S6+ 2H]+ / 2: 1092.1, found: 1092.2.

[0126] While this invention has been particularly shown and described with references toexample embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.11Tag-2-3'-dG(iBu)-dC(Bz)-dG(iBu)-5'-OH

[0112] To a stirred solution of DMT-dG(iBu) amidite (0.27 g, 0.33 mmol, 1.5 eq) inDCM (2.5 rnL) was added 10 (0.46 g, 0.22 mmol, 1 eq) as a solution in DCM (2.5 mL). To the resulting solution was further added 5 mL of DCM and the solution was distilled under vacuum at 30 °C to azeotropically dry the solution. This azeotrope cycle was performed two additional times and concentrated down to 5 mL total solution. To this was added powdered activated 3 A molecular sieve (0.46 g, 100 wt%). The mixture was stirred at RT for 1 h. DCI (0.039 g, 0.33 mmol, 1.5 eq) was added to the reaction and continued stirring for 3 h. To the reaction was added xanthane hydride (0.050 g, 0.33 mmol, 1.5 eq). The reaction continued stirring at RT for 3 h. The IPC was analyzed by31P-NMR which showed the complete conversion. The reaction mixture was filtered, and the filtrate was concentrated down to dryness. To this was added DCM (5 mL) and powdered activated 3 A molecular sieve (0.42 g). The mixture was stirred for 45 minutes. To the reaction wasthen added 1 -dodecanethiol (0.17 mL, 0.72 mmol, 3.3 eq). The reaction was cooled to 0 ±5 °C and TFA (0.17 mL, 2.18 mmol, 10 eq) was added dropwise. The resulting reddish reaction mixture continued stirring for 3 h. The reaction was quenched with the addition of NMI (0.19 mL, 0.24 mmol, 11 eq). The mixture was filtered, and the filtrate was further washed with 10 wt% aq. K2CO3 (5 mL) followed by brine (4 mL x 2). The resulting organic layer was dried over anhydrous MgSCL, filtered, and concentrated in vacuo. The residue was dissolved to a minimum of DCM (1 mL). The solution was added to the stirred MeCN (20 mL) at RT. The resulting slurry was stirred for 1 h, filtered and the solid was washed with MeCN (5 mL x 2). The solid was dried under vacuum at RT for 0.5 h. The light-yellow solid of 11 was obtained in 0.49 g. The sample was slightly impure with unreacted starting material. Therefore, a second cycle of detritylation was performed and the light-yellow solid was isolated in 0.39 g (69%).31P-NMR (CDCh, 242.9 MHz) 66.31.

[0113] 10 mg of the solid 11 was treated with NH4OH (0.4 mL, 28-30%) and the mixture was stirred at 65 °C for 3 h. An aliquot was diluted with MeCNThO (1 : 1) and the mixture was filtered (0.2 pm filter). The filtrate was analyzed by LCMS. MS (ESI+): m / z calcd for5’-HO-GCG-OH-3’ [C29H37N13O14P2S2 + H]+: 918.2, found: 918.2.

[0114] Tag-3'-dG(iBu)-dC(Bz)-dG(iBu)-dT-5'-OH (12)11Tag-2-3'-dG(iBu)-dC(Bz)-dG(iBu)-5'-OH

[0115] To a stirred solution of DMT-dT amidite (0.13 g, 0.18 mmol, 1.5 eq) in DCM (2.5 mL) was added 11 (0.34 g, 0. 12 mmol, 1 eq) as a solution in DCM (2.5 mL). To the resulting solution was added further 5 mL of DCM was added and the solution was distilled under vacuum at 30 °C to azeotropically dry the solution. This azeotrope cycle was performed two additional times and concentrated down to 4 mL total solution. To this was added powdered activated 3 A molecular sieve (0.34 g, 100 wt%). The mixture was stirred at RT. After 1 h, DCI (0.021 g, 0.18 mmol, 1.5 eq) was added to the reaction and continued stirring. The HPLC analysis of the IPC showed the completion of reaction after 2 h. To the reaction was added xanthane hydride (0.020 g, 0.13 mmol, 1.1 eq). The reaction continued stirring at RT. After overnight stirring at RT, IPC was analyzed by 31P- NMR which showed the complete conversion. To the reaction was then added 1-dodecanethiol (0.09 mL, 0.39 mmol, 3.3 eq). The reaction was cooled to 0 ± 5 °C and TFA (0.09 mL, 1.2 mmol, 10 eq) was added dropwise. The resulting reddish reaction mixture continued stirring at 0 ± 5 °C for 2 h. The reaction was quenched with the addition of NMI (0.15 mL, 1.8 mmol, 15 eq). The mixture was filtered, and the filtrate was further washed with 10 wt% aq. K2CO3 (5 mL) followed by brine (4 mL x 2). The resulting organic layer was dried over anhydrous MgSCL, filtered, and concentrated in vacuo. The residue was dissolved to a minimum of DCM (1 mL). The solution was added to the stirred MeCN (20 mL) at RT. The resulting slurry was stirred for 1 h, filtered and the solid was washed with MeCN (5 mL x 2). The solid was dried under vacuum at RT for 1 h. The light-yellow solid 12 was isolated in 0.32 g (91%).31P-NMR (CDCh, 242.9 MHz) 66.52, 66.33, 66.21, 66.13

[0116] 10 mg of the solid 12 was treated with NH4OH (0.4 mL, 28-30%) and the mixture was stirred at 65 °C for 3 h. An aliquot was diluted with MeCNTEO (1 : 1) and the mixture was filtered (0.2 pm filter). The filtrate was analyzed by LCMS. MS (EST): m / z calcd for 5’-HO-TGCG-OH-3’ [C39H50N15O20P3S3 - H]’: 1236.2, found: 1236.3.

[0117] Tag-3'-dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-5'-QH (13)12Tag-2-3'-dG(iBu)-dC(Bz)-dG(iBu)-dT-5'-OH13Tag-2-3'-dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-5'-OH

[0118] To a stirred solution of DMT-dT amidite (0.11 g, 0.15 mmol, 1.5 eq) in DCM (2.5 mL) was added 12 (0.30 g, 0. 10 mmol, 1 eq) as a solution in DCM (2.5 mL). The solution was distilled under vacuum at 30 °C to azeotropically dry the solution. This azeotrope cycle was performed two additional times and concentrated down to 4 mL total solution. To this was added powdered activated 3 A molecular sieve (0.32 g, 106 wt%). Themixture was stirred at RT. After 1 h, DCI (0.018 g, 0. 15 mmol, 1.5 eq) was added to the reaction and continued stirring. The HPLC analysis showed the completion of reaction after 3 h. To the reaction was added xanthane hydride (0.017 g, 0.11 mmol, 1.1 eq). The reaction continued stirring at RT. After 2.5, IPC was analyzed by 31P-NMR which showed the complete conversion. The reaction mixture was filtered. To the filtrate was then added 1 -dodecanethiol (0.08 rnL, 0.34 mmol, 3.3 eq). The reaction was cooled to 0 ± 5 °C and TFA (0.08 mL, 1.02 mmol, 10 eq) was added dropwise. The resulting reddish reaction mixture was continued stirring at 0 ± 5 °C for 2 h. The reaction was quenched with the reverse addition to 10 wt% aq. K2CO3 (5 mL). The organic layer was washed with brine (4 mL x 2). The resulting organic layer was dried over anhydrous MgSCfi. filtered, and concentrated in vacuo. The residue was dissolved to a minimum of DCM (1 mL). The solution was added to the stirred MeCN (15 mL) at RT. The resulting slurry was stirred for 1 h, filtered and the solid was washed with MeCN (5 mL x 2). The solid was dried under vacuum at RT for 1 h. The light-yellow solid of 13 was isolated in 0.24 g (71%).31P-NMR (CDCh, 242.9 MHz) 66.54, 66.32, 66.27, 66.16, 66.12.

[0119] 10 mg of the solid 13 was treated with NH4OH (0.4 mL, 28-30%) and the mixture was stirred at 65 °C for 6 h. An aliquot was diluted with MeCN / LfiO (1 : 1) and the mixture was filtered (0.2 pm filter). The filtrate was analyzed by LCMS. MS (ESI+): m / z calcd for 5’-HO-TTGCG-OH-3’ [C49H63N17O26P4S4 + H]+: 1558.2, found: 1558.3.

[0120] Tag-2-3'-dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-dC(Bz)-5'-QH (14)13Tag-2-3'-dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-5'-OH14Tag-2-3' -dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-dC(Bz)-5'-0H

[0121] To a reaction vial was charged DMT-dC(Bz) amidite (0.08 g, 0.09 mmol, 1.5 eq), 13 (0.20 g, 0.06 mmol, 1 eq), powdered activated 3 A molecular sieve (0.25 g, 125 wt%) and DCM (3.0 mL). The resulting mixture was stirred at RT. After 1 h, DCI (0.011 g, 0.09 mmol, 1.5 eq) was added to the reaction and continued stirring at RT. After 3.5 h Xanthane hydride (0.010 g, 0.07 mmol, 1.1 eq) was added to the reaction. The reaction continuedstirring at RT overnight. The IPC analyzed by 31P-NMR showed the complete conversion. To the reaction was then added 1 -dodecanethiol (0.05 mL, 0.20 mmol, 3.3 eq). The reaction was cooled to 0 ± 5 °C and TFA (0.05 mL, 0.60 mmol, 10 eq) was added dropwise. The resulting reddish reaction mixture continued stirring at 0 ± 5 °C for 2 h. The reaction was quenched with the addition of NMI (0.06 mL, 0.66 mmol, 11 eq). The mixture was filtered, and the filtrate was further washed with 10 wt% aq. K2CO3 (5 mL). The organic layer was washed with brine (4 mL x 2). The resulting organic layer was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was dissolved to minimum DCM (0.5 mL). The solution was added to the stirred MeCN (10 mL) at RT. The resulting slurry was stirred for 1 h, filtered and the solid was washed with MeCN (2 mL x 2). The solid was dried under vacuum at RT for 1 h. The light-yellow solid of 14 was isolated in 0.19 g (83%). 31P-NMR (CDC13, 242.9 MHz) 66.54, 66.32, 66.27, 66.16, 66.12.

[0122] 10 mg of the solid 14 was treated with NH4OH (0.4 mL, 28-30%) and the mixture was stirred at 65 °C for 6 h. An aliquot was diluted with MeCNTTO (1 : 1) and the mixture was filtered (0.2 pm filter). The filtrate was analyzed by LCMS. MS (ESI+): m / z calcd for 5’-HO-CTTGCG-OH-3’ [C58H75N20O31P5S5 + H]+: 1863.2, found: 1863.2.

[0123] Tag-2-3'-dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-dC(Bz)-dT-5'-OH (15)14Tag-2-3'-dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-dC(Bz)-5'-OH15Tag-2-3'-dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-dC(Bz)-dT-5'-OH

[0124] To a reaction vial was charged DMT-dT amidite (0.05 g, 0.06 mmol, 1.5 eq), 14(0.16 g, 0.04 mmol, 1 eq) and DCM (10 mL). The solution was distilled under vacuum at 30 °C to azeotropically dry the solution and concentrated down to 2 mL total solution. Tothe vial was added powdered activated 3 A molecular sieve (0.16 g, 100 wt%). The mixture was stirred at RT. After 1 h, DCI (0.007 g, 0.06 mmol, 1.5 eq) was added to the reaction and continued stirring for 2 h. To the reaction was added xanthane hydride (0.007 g, 0.05 mmol, 1.1 eq). The reaction continued stirring at RT overnight. IPC was analyzed by 31P- NMR which showed the complete conversion. To the reaction was then added 1- dodecanethiol (0.03 mL, 0.14 mmol, 3.3 eq). The reaction was cooled to 0 ± 5 °C and TFA (0.03 mL, 0.41 mmol, 10 eq) was added dropwise. The resulting reddish reaction mixture continued stirring at 0 ± 5 °C for 2 h. The reaction was quenched with the addition of NMI (0.04 mL, 0.44 mmol, 11 eq). The mixture was filtered, and the filtrate was further washed with 10 wt% aq. K2CO3 (2.5 mL). The organic layer was washed with brine (2 mL x 2). The resulting organic layer was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was dissolved to minimum DCM (0.5 mL). The solution was added to the stirred MeCN (5.5 mL) at RT. The resulting slurry was stirred for 1 h, filtered and the solid was washed with MeCN (2 mL x 2). The solid was dried under vacuum at RT for 1 h. The light-yellow solid of 15 was isolated in 0.13 g (78%). 31P-NMR (CDC13, 242.9 MHz) 66.54, 66.33, 66.17.

[0125] 5 mg of the solid 15 was treated with NH4OH (0.2 mL, 28-30%) and the mixture was stirred at 65 °C for 5 h. An aliquot was diluted with MeCN / LLO (1 : 1) and the mixture was filtered (0.2 pm filter). The filtrate was analyzed by LCMS. MS (ESI+): m+z / z calcd for 5’-HO-TCTTGCG-OH-3’ [CesH^CLvPeSe + 2H | / 2: 1092.1, found: 1092.2.

[0126] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

-34- PCT 87311-3013 (P101781WO01) CLAIMS 1. A selectively soluble support compound represented by Formula (I):, whereinR1 is a R2 is H or a straight or branched C1-C40 aliphatic group; R3 is H or C1-C40 alkyl; and Y is selected from the group consisting of H, succinate, -C(O)-X, -C1-C10 alkyl- C(O)-X, -C(O)-(C1-C10 alkyl)-C(O)-X, and -C(O)-aryl-C(O)-X, wherein X is Cl, Br, I, F, imidazole, or another leaving group.

2. The selectively soluble support compound of claim 1, wherein Y is H.

3. The selectively soluble support compound of claim 2, wherein R2 is a methyl group.

4. The selectively soluble support compound of claim 3, wherein R1 is C18H37.

5. The selectively soluble support compound of claim 1, wherein R1 is C18H37.

6. The selectively soluble support compound of claim 5, wherein R3 is the C1-C40alkyl.

7. The selectively soluble support compound of claim 6, wherein R3 is methyl.

8. The selectively soluble support compound of claim 4, wherein R3 is H.

9. The selectively soluble support compound of claim 1, wherein Y is selected fromthe group consisting of -COOH, -C(O)-X, and -O-C(O)-X; and X is imidazole.

10. The selectively soluble support compound of claim 1, wherein the compound isO Me N C18H37O Me O NH N N C18H37N H Me N O O C18H37.-35- PCT 87311-3013 (P101781WO01) 11. A liquid phase method of producing an oligonucleotide comprising a step ofgroup of a nucleoside or support in at least one location selected 3′-position, 5′-position and a nucleobase or a 3′-hydroxyl group, to H-phosphonation hydroxyl group into an H-phosphonatedis a compound represented by Formula (I): O R2N R1O Y R2O N N N R1N R2N R3O R1O (I), wherein R1 is a R2is H or a R3 is H or Y is selected from C10alkyl-C(O)-X, - C(O)-(C1-C10 Br, I, F, imidazole, or another leaving12. The method of claim 11, wherein Y is selected from the group consisting of -COOH, -C(O)-X, and -O-C(O)-X; and X is imidazole.

13. The method of claim 11, wherein the selectively soluble support compoundO Me N C18H37O Me O NH N N C18H37N H Me N O O represented by Formula (I) is C18H37.-36- PCT 87311-3013 (P101781WO01) 14. The method of claim 11, which comprises at least one elongation reaction cyclecomprising a step of a selectively soluble support from the group consisting of 2′- moiety and having a 5′-hydroxyl to convert the 5′-hydroxyl group form.

15. The method of claim 14, wherein the elongation reaction cycle comprises:a first step including deprotecting a first nucleoside or first oligonucleotide having a selectively soluble support represented by Formula (I) in at least one location selected from the group consisting of 2′-position, 3′-position and a nucleobase moiety, having a 3′- hydroxyl group protected with a basic protecting group or a selectively soluble support represented by Formula (I), and having a 5′-hydroxyl group protected with a temporary protecting group, to remove the temporary protecting group to form a 5′-hydroxyl group, a second step including converting the resultant 5′-hydroxyl group into an H- phosphonated form using an H-phosphonate reagent, and a third step of forming an oligomer of the first nucleoside or first oligonucleotide with a second nucleoside or second oligonucleotide having a 3′-hydroxyl group and having a 5′-hydroxyl group protected with a temporary protecting group, by forming a phosphite diester bond from the 5′-hydroxyl group, now converted to the H-phosphonated form, of the first nucleoside or first oligonucleotide and the 3′-hydroxyl group of the second nucleoside or second oligonucleotide.

16. The method of claim 15, further comprising a fourth step including converting thephosphite diester bond of the oligomer into a phosphodiester bond, a thiophosphodiester bond, a boranophosphodiester bond, an aminophosphodiester bond, a phosphodiester bond protected with a basic protecting group, or a thiophosphodiester bond protected with a basic protecting group.-37- PCT 87311-3013 (P101781WO01) 17. The method of claim 15, further comprising a fourth step including converting thephosphite diester bond of the oligomer into a phosphodiester bond, a thiophosphodiester bond, a boranophosphodiester bond or an aminophosphodiester bond.

18. The method of claim 17, further comprising a fifth step of adding a polar solvent toa reaction mixture obtained from at least one step selected from the group consisting of the first step to the fourth step to form a precipitate, and collecting the precipitate by solid liquid separation.

19. The method of claim 18, further comprising a step of cleaving the formedoligonucleotide from the compound of Formula (I).

20. The method of claim 11, further comprising a step of cleaving the formed peptidefrom the compound of Formula (I).

17. The method of claim 15, further comprising a fourth step including converting the phosphite diester bond of the oligomer into a phosphodiester bond, a thiophosphodiester bond, a boranophosphodi ester bond or an aminophosphodiester bond.

18. The method of claim 17, further comprising a fifth step of adding a polar solvent to a reaction mixture obtained from at least one step selected from the group consisting of the first step to the fourth step to form a precipitate, and collecting the precipitate by solid liquid separation.

19. The method of claim 18, further comprising a step of cleaving the formed oligonucleotide from the compound of Formula (I).

20. The method of claim 11, further comprising a step of cleaving the formed peptide from the compound of F ormul a (I) .

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