Selectively soluble 5-hydroxyisophthalic acid-derived supports for liquid-phase oligonucleotide and peptide synthesis

Abstract

The present invention provides compounds useful as selectively soluble supports (or tags) for oligonucleotide and peptide 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) or Formula (II), which is derived from 5-hydroxyisophthalic acid.

Description

(P101782W001)SELECTIVELY SOLUBLE 5-HYDROXYISOPHTHALIC ACID-DERIVED SUPPORTS FOR LIQUID-PHASE OLIGONUCLEOTIDE AND PEPTIDE SYNTHESISCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 690,616 filed on September 4, 2024, which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] The field of the disclosure relates generally to amino acids, peptides, proteins, and nucleic acids for pharmaceutical industry and, more specifically, using selectively soluble supports for liquid phase organic synthesis of peptides and oligonucleotides.BACKGROUND OF THE INVENTION

[0003] The present invention relates to selectively soluble supports for liquid-phase oligonucleotide and peptide 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 and peptide synthesis, particularly liquid phase synthesis, and methods of synthesis using such a compound.

[0004] Despite the widespread adoption of solid-phase synthesis of oligonucleotides and peptide due to 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.(P101782W001)

[0005] Conversely, liquid-phase synthesis on a soluble support offers a compelling alternative for scaling up oligonucleotide and peptide 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 and peptides.

[0006] Additionally, the liquid-phase approach can inherently accommodate larger reaction volumes without the physical constraints imposed by solid supports, thus enabling the synthesis of oligonucleotides and peptides 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.

[0007] In an attempt to perform reactions in a homogeneous liquid phase while utilizing the 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.

[0008] A particular component dissolved in a solution can be precipitated only when predetermined 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 and peptide 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.(P101782W001)

[0009] To solve such problems, a method using a carrier molecule wherein a dissolved state 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.

[0010] Soluble supports for oligonucleotide and peptide synthesis have been engineered to enhance the efficiency and scalability of the synthesis process. P-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.

[0011] Still there is a need in the art for new selectively soluble supports for liquid phase organic synthesis of oligonucleotides and peptides which are superior in broad utility and stability, that are useful as a protecting group (anchor) for oligonucleotide and peptide synthesis in the liquid phase.BRIEF SUMMARY OF THE INVENTION

[0012] In one aspect, disclosed herein is selectively soluble support compound represented by Formula (I):(P101782W001)wherein,Ri is a straight or branched C6-C40 aliphatic group;R2 is H or a straight or branched C1-C40 aliphatic group; andY is selected from the group consisting of H, succinate, -C(O)-X, -C1-C10 alkyl- C(O)-X, -C(0)-(Ci-Cio alkyl)-C(O)-X, and -C(O)-aryl-C(O)-X. wherein X is Cl, Br, I, F, imidazole, or other leaving group.

[0013] Compounds of Formula (I) can be used as both a selectively soluble support for oligonucleotide 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.

[0014] Compounds which can be protected by a compound of Formula (I) may be any compound having a functional group such as a carboxy 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.

[0015] 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(P101782W001) 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):(I), wherein, Ri is a straight or branched C6-C40 aliphatic group; R2 is H or a straight or branched C1-C40 aliphatic group; and Y is selected from the group consisting of H, succinate. -C(O)-X. -, -C1-C10 alkyl- C(O)-X, -C(0)-(Ci-Cio alkyl)-C(O)-X, and -C(O)-aryl-C(O)-X, wherein X is Cl, Br, I, F, imidazole, or other leaving group.

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

[0017] In another aspect, disclosed herein is selectively soluble support compound represented by Formula (II):wherein Ri is a straight or branched C6-C40 aliphatic group; R2 is H or a straight or branched C1-C40 aliphatic group; R3 is H or -O-alkyl; R4 is H or -O-alkyl; and Y is selected from the group consisting of -OH, -COOH, -O-C(O)-X. wherein X is Cl, Br, I, F, imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralky l group is a straight or branched C1-C30 alkyl.(P101782W001)

[0018] Compounds of Formula (II) can be used as both a support for peptide 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 the carbonyl group is protected by a compound of Formula (II) is characterized by tending to be in a liquid state and its high solubility7in a solvent. Accordingly, a compound in which a functional group is protected by a compound of Formula (II) 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.

[0019] Compounds which can be protected by a compound of Formula (II) may be any compound 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.

[0020] The embodiments and features of the present invention can be used alone or in combinations with each other.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings.

[0022] FIG. 1 is anXH NMR (600 MHz, CD3OD) spectrum of compound 2 of the Example 1 ;

[0023] FIG. 2 is anXH NMR (600 MHz, CDCI3) spectrum of compound 3 of the Example 1;

[0024] FIG. 3 is anXH NMR (600 MHz, CDCI3) spectrum of compound 4 of the Example 1;

[0025] FIG. 4 is anXH NMR (600 MHz, CDCI3) spectrum of compound 8 of the Example 1;(P101782W001)

[0026] FIG. 5 is an1H NMR (600 MHz, CDCh) spectrum of compound 9 of the Example 1;

[0027] FIG. 6 is an 'H NMR (600 MHz, CDCh) spectrum of compound 7 of the Example 1;

[0028] FIG. 7 is anXH NMR (600 MHz, CDCh) spectrum of compound 11 of the Example 2:

[0029] FIG. 8 is anXH NMR (600 MHz, CDCh) spectrum of compound 12 of the Example 2;

[0030] FIG. 9 is a31P NMR (600 MHz, CDCh) spectrum of compound 12 of the Example 2;

[0031] FIG. 10 is anXH NMR (600 MHz, CDCh) spectrum of compound 13 of the Example 2;

[0032] FIG. 1 1 is a31P NMR (600 MHz, CDCh) spectrum of compound 13 of the Example 2;

[0033] FIG. 12 is anXH NMR (600 MHz, CDCh) spectrum of compound 16 of the Example 2;

[0034] FIG. 13 is anXH NMR (600 MHz, CDCI3) spectrum of compound 17 of the Example 2;

[0035] FIG. 14 is a3'P NMR (600 MHz, CDCh) spectrum of compound 17 of theExample 2:

[0036] FIG. 15 is anXH NMR (600 MHz, CDCh) spectrum of compound 18 of the Example 2;

[0037] FIG. 16 is anXH NMR (600 MHz, CDCh) spectrum of compound 19 of the Example 2;

[0038] FIG. 17 is anXH NMR (600 MHz, CDCh) spectrum of compound 20 of the Example 2;

[0039] FIG. 18 is a31P NMR (600 MHz. CDCh) spectrum of compound 20 of the Example 2;(P101782W001)

[0040] FIG. 19 is an 'H NMR (600 MHz, CDCh) spectrum of compound 21 of the Example 2;

[0041] FIG. 20 is a31P NMR (600 MHz, CDCh) spectrum of compound 21 of the Example 2;

[0042] FIG. 21 is anXH NMR (600 MHz, CDCh) spectrum of compound 22 of the Example 2:

[0043] FIG. 22 is a31P NMR (600 MHz. CDCI3) spectrum of compound 22 of the Example 2;

[0044] FIG. 23 is an 'H NMR (400 MHz, CDCh) spectrum of compound 25 of the Example 3;

[0045] FIG. 24 is an 'H NMR (400 MHz, CDCh) spectrum of compound 26 of the Example 3;

[0046] FIG. 25 is an LCMS (ES+) chromatograph of compound 26 of the Example 3;

[0047] FIG. 26 is an1H NMR (400 MHz, CDCh) spectrum of compound 27, Tag 5P-S of the Example 3;

[0048] FIG. 27 is an ’H NMR (400 MHz, CDCh) spectrum of compound 29 of the Example 4;

[0049] FIG. 28 is an LCMS (ES+) chromatograph of compound 29 of the Example 4;

[0050] FIG. 29 is an1H NMR (400 MHz, CDCh) spectrum of compound 30 of the Example 4;

[0051] FIG. 30 is an LCMS (ES+) chromatograph of compound 30 of the Example 4;

[0052] FIG. 31 is anXH NMR (400 MHz, CDCh) spectrum of compound 31 of the Example 4;

[0053] FIG. 32 is anlH NMR (400 MHz, CDCh) spectrum of compound 32 of the Example 4;

[0054] FIG. 33 is an LCMS (ES+) chromatograph of compound 32 of the Example 4;

[0055] FIG. 34 is an 'H NMR (400 MHz, D2O) spectrum of hexamer 33 of the Example 4; and(P101782W001)

[0056] FIG. 35 is an LCMS (ES+) chromatograph of hexamer 33 of the Example 4.DETAILED DESCRIPTION OF THE INVENTION

[0057] Unless otherw ise specified in the sentences, any technique terms and scientific terms 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.Definitions

[0058] As used herein, the terms "a", "an", "the" and the like refer to both the singular and plural unless the context clearly indicates otherwise. "A bottle", for example, refers to a single bottle or more than one bottle.

[0059] 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.

[0060] 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.

[0061] As used herein, the term “alkyl” means a saturated hydrocarbon group which is straight-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(P101782W001) 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, neopenty l), hexyl, isohexyl, heptyl, octyl, nonyl, 4,4-dimethylpentyl, 2,2,4-trimethylpentyl, decyl, undecyl, dodecyl, 2-methyl-l- propyl, 2-methyl-2 -propyl, 2-methyl-l -butyl, 3-methyl-l-butyl, 2-methyl-3-butyl, 2- methyl-1 -pentyl, 2.2-dimethyl-l -propyl, 3 -methyl- 1 -penty l, 4-methyl-l -pentyl. 2-methyl-2- pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2.2-dimethyl-l -butyl, 3,3-dimethyl-l-butyl, 2-ethyl-l -butyl, and the like.

[0062] As used herein, the term “alkylamino” means an amino group substituted by at least one alkyd group. In some embodiments, the alkyl group is a lower alkyl group having from 1 to 6 carbon atoms. Alkydamino groups include, but are not limited to, -NHCH2CH3, -NH(CH2)2CH3, -NH(CH2)3CH3, -NH(CH2)4CH3, and -NH(CH2)5CH3, and the like.

[0063] As used herein, the term “amino’' means -NH2.

[0064] As used herein, the term “ary l” 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, pheny l, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and tetrahydronaphthyl, and the like.

[0065] As used herein, the term “aralkyl” or “arylalkyl” refers to a monovalent alkyl group substituted with one or more ary 1 groups. In certain embodiments, the aralky l 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-pheny Ipropyl. In certain embodiments, the aralky 1 is optionally substituted with one or more.

[0066] The term “halogen”, “halide” or “halo” refers to fluorine, chlorine, bromine, and / or iodine.

[0067] As used herein, the term “nucleoside” is the constituent unit of an oligonucleotide means a compound wherein a nucleic acid base is bonded to the T-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(P101782W001) 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:

[0068] As used herein, the term “nucleotide” means a compound wherein a phosphoric acid group is bonded to a nucleoside.

[0069] As used herein, the term “oligonucleotide” means a compound wherein one or more 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).

[0070] As used herein, the term “nucleoside'’ and “nucleotide” encompass morpholino nucleoside and morpholino nucleoside having a morpholine residue instead of a ribose residue or a deoxyribose residue.

[0071] The terms “linker” and “linkage” may be used interchangeably and mean a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches.(P101782W001) or is attached to, for example, a selectively soluble support disclosed herein to a nucleoside or an oligonucleotide.

[0072] Also as used herein, the description of one or more method steps does not preclude 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.

[0073] Where a range of numbers is presented in the application, it is understood that the range 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

[0074] The compounds of the present invention are selectively soluble support compounds represented by Formula (I):'(| ) wherein,Ri is a straight or branched C6-C40 aliphatic group;R2 is H or a straight or branched C1-C40 aliphatic group; andY is selected from the group consisting of H, succinate, -C(O)-X, -C1-C10 alkyl- C(O)-X, -C(0)-(Ci-Cio alkyl)-C(O)-X, and -C(O)-aiyl-C(O)-X. wherein X is Cl, Br, I, F, imidazole, or other leaving group.

[0075] A compound represented by Formula (I) of the present invention is bonded to a compound 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(0)-(Ci-Cio(P101782W001) 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.

[0076] The “aliphatic group’' of Ri is an aliphatic hydrocarbon group consisting of straight 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.

[0077] The “aliphatic group’' of R2 is an aliphatic hydrocarbon group consisting of straight 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.

[0078] Examples of the “aliphatic group” include monovalent groups such as an alkyl group, 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 lert-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, Ri is a stearyl group, C18H37.

[0079] In some embodiments, Y is H and Ri is C18H37. In other embodiments, Y is succinate and Ri is C18H 7. In yet other embodiments, Y is H or succinate, and R2 is H.

[0080] In some embodiments, Y is H, R2 is a methyl group, and Ri is C18H37. In other embodiments, R2 is methyl. In yet other embodiments, R2 is a straight or branched C1-C40 aliphatic group, such as, for example, a straight C18H37 chain.(P101782W001)

[0081] In some embodiments, Y is succinate. In other embodiments, Y is selected from the group consisting of -C(O)-X. -Ci-Cio alkyl-C(O)-X, -C(0)-(Ci-Cio alkyl)-C(O)-X, and -C(O)-aryl-C(O)-X; and X is imidazole. In yet other embodiments. Y is selected from the group consisting of -C(O)-X, -Ci-Cio alkyl-C(O)-X, -C(0)-(Ci-Cio alkyl)-C(O)-X, and - C(O)-aiyl-C(O)-X; and X is selected from the group consisting of Cl, Br, I, and F.

[0082] Examples of a compound of Formula (I) include the compounds N 1 ,N3- dioctadecyl-5-((6-oxo-6-(piperazin-l-yI)-hexyI)oxy)isophthalamide,(piperazin- 1 -yl)-hexyl)oxy )isophthalamide,

[0083] The compounds of the present invention are selectively soluble support compounds represented by Formula (II):wherein,Ri is a straight or branched C6-C40 aliphatic group:R2 is H or a straight or branched C1-C40 aliphatic group;R3 is H or -O-alkyl;R4 is H or -O-alkyl; andY is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F, imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or(P101782W001) branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7- C30 aralkyl group is a straight or branched C1-C30 alkyl.

[0084] A compound represented by Formula (II) of the present invention is bonded to a compound to be supported and protected via group Y. That is, the compound of the present invention wherein Y is a -OH, -COOH, O-C(O)-X, wherein X is Cl, Br, I, F, imidazole or a leaving group, or an -NHR group is bonded to the C-terminal of, for example, an amino acid or a peptide or an oligonucleotide for protection thereof.

[0085] The “aliphatic group’’ of Ri is an aliphatic hydrocarbon group consisting of straight 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.

[0086] The “aliphatic group’’ of R2 is an aliphatic hydrocarbon group consisting of straight 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.

[0087] Examples of the “aliphatic group” include monovalent groups such as an alkyl group, 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-but l 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 steary 1 group, an aralkyl group, a behenyl group, an oleyl group, an isostearyl group and the like and divalent groups derived therefrom. In preferred embodiments, Ri is a stearyl group, C18H37.(P101782W001)

[0088] As R, a hydrogen atom, a C 1-6 alkyl group or a C7-16 aralkyl group is preferable, a hydrogen atom, methyl, ethyl or benzyl is more preferable, and a hydrogen atom is particularly preferable.

[0089] In some embodiments, Y is -OH. In other embodiments, Y is -OH and Ri is C18H37.

[0090] In other embodiments, Y is -NHR. In embodiments when Y is -NHR. R is H. a C1-C30 alkyl group, or a C7-C30 aralkyl group. In embodiments wherein R is C1-C30 alkyl, R is preferably methyl. In embodiments wherein R is C7-C30 aralkyl, R is preferably methyl.

[0091] In some embodiments, R is H.

[0092] Examples of the “aralkyl group” for R include a C7-30 aralkyl group. Preferred is a C7-20 aralkyl group, more preferred is a C7-I6 aralkyl group (a Ce-40 aryl-Ci-6 alkyl group). Preferable examples include benzyl, 1 -phenylethyl, 2-phenylethyl, 1 -phenylpropyl, naphthylmethyl, 1 -naphthylethyl, 1 -naphthylpropyl, and the like, and particularly preferred is benzyl.

[0093] In some embodiments, Rs and R4 are not the same. In some embodiments, R3 is H and R4 is O-alkyl. In some embodiments, R3 is O-alkyl and R4 is H. In other embodiments, R3 is O-methyl and R4 is O-ethyl.

[0094] In some embodiments of Formula (II), wherein Ri is C18H37. R2 is H, R3 is -OMe, R4 is H, and Y is -OH, an example of a selectively soluble support compound isAnother example of the selectively soluble support compound of Formula (II), wherein Ri is C18H37, R2 is H, R3is H, R4 is -(P101782W001)Production Method of Compounds of Formula (I)

[0095] The compounds represented by Formula (I) of the present invention can be made by any method known to those skilled in the art. An exemplary method is detailed inExample 1 for the compound,Production Method of Compounds of Formula (ID

[0096] The compounds represented by Formula (II) of the present invention can be made by any method known to those skilled in the art. An exemplary method is detailed in Example 3 for the compound, which is a compound of Formula (I) wherein Y is -OH and each of Ri and R2 are C18H37.Use as a Selectively Soluble Support in Organic Synthesis of Oligonucleotides

[0097] Next, an oligonucleotide production method according to the present invention will 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-iner 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(0)-(Ci-Cio 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(P101782W001) n is 1, the n-mer oligonucleotide is understood as a nucleoside. The same applies to the p- mer oligonucleotide.

[0098] The oligonucleotide production method includes a step of subjecting a nucleoside or 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.

[0099] Further, the oligonucleotide production method includes at least one elongation reaction cycle including the above H-phosphonation step.

[0100] The oligonucleotide production method is characterized in that the elongation reaction 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).

[0101] Step (a)

[0102] Step (a) is a temporary protecting group removal step including deprotecting a nucleoside 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.

[0103] Step (b)

[0104] Step (b) is a phosphonation step including converting the hydroxyl group resulting from the removal of the temporary protecting group into an H-phosphonated form using an H-phosphonate reagent.

[0105] Step (c)(P101782W001)

[0106] Step (c) is a coupling step including adding a nucleoside or oligonucleotide having a 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.

[0107] Step (d)

[0108] Step (d) is a conversion step including converting the phosphite diester bond into a phosphodiester bond, a thiophosphodiester bond, an aminophosphodi ester 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.

[0109] Here, the amino group in the aminophosphodiester bond is unsubstituted or is substituted with one or two Ci-6 alkyl groups.

[0110] The phosphodiester bond protected with a basic protecting group is a bond formed by substituting a phosphodi ester bond with, for example, a C1-40 alkyl group, a C3-6 cycloalkyl group, a Ce-io aryl 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 C1-6 alkyl group is unsubstituted, or is substituted with a substituent such as a halogen atom or a cyano group. The C3-6 cycloalkyl group, the Ce-io 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.

[0111] The thiophosphodiester bond protected with a basic protecting group is a bond formed by substituting a thiophosphodi ester bond with, for example, a C1-40 alkyl group, a C3-6 cycloalkyl group, a Ce-io aryl 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 C1-40 alkyl group is unsubstituted, or is substituted with a substituent such as a halogen atom or a cyano group. The C3-6 cycloalkyl group, the Cg-io 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.(P101782W001)

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] Step (e)

[0117] Step (e) is a separation step of adding a polar solvent to the reaction mixture obtained 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.

[0118] 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, dimethyl acetamide, 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.

[0119] Step (e) is specific to the liquid phase synthesis method using a selectively soluble support 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.(P101782W001)

[0120] The number of Steps (e) present in the elongation reaction cycle is not particularly limited. Step (e) may be performed after any of Step (a) to Step (d).

[0121] 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).

[0122] The elongation reaction cycle preferably includes one to four Steps (e). To manage 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.

[0123] In another embodiment, it is more preferable that Step (e) be performed one time after 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.

[0124] If the circumstances permit the amount of byproducts to be controlled by controlling 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.

[0125] The oligonucleotide production method may further include Step (f), in which case the desired oligonucleotide can be isolated and produced.

[0126] Step (f)

[0127] Step (I) 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).

[0128] In a final step (excluding any purification steps), the formed oligonucleotide is cleaved 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.(P101782W001)

[0129] Specific examples of liquid phase oligonucleotide production with partially soluble 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.Use as a Selectively Soluble Support in Organic Synthesis for Peptides

[0130] The peptide synthesis method of the present invention can be suitably used in a peptide production method using a selectively soluble support (i.e., a carrier or Tag) capable of reversibly repeating the dissolved state and the insolubilized state.

[0131] As the peptide production method including the peptide synthesis method of the present invention, for example, steps as shown below are exemplified.

[0132] Compounds which can be protected by a compound of Formula (II) may be any compound 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.

[0133] The compound of the present invention can be used as a reagent to protect a carboxyl group and the like, that is, a protecting reagent for amino acid or peptide, in organic synthesis reactions, preferably peptide synthesis and the like. Specifically, it is preferably introduced as a protecting group (anchor) of C-terminal functional groups such as a carboxyl group, a carboxamide group, a thiol group and the like, and side chain functional groups (hereinafter to be referred to as C-terminal etc.). When using as a protecting reagent, the compound may be activated to allow reaction with a substituent to be protected or may be converted to an equivalent to allow reaction.

[0134] The method of synthesizing a peptide using a compound of Formula (II) as a protecting agent includes, for example, the following steps (1) to (4). In this peptide synthesizing method, the protected peptide obtained in each step and the target peptide can be separated by liquid-liquid separation.

[0135] step (1) a step of dissolving the compound of the present invention in soluble solvent (dissolution step),(P101782W001)

[0136] step (2) a step of bonding the compound of the present invention dissolved in soluble solvent as obtained in the above-mentioned step to a reaction substrate (bonding step).

[0137] step (3) a step of precipitating the bonded product obtained in the above- mentioned step (precipitation step), and

[0138] step (4) a step of redissolving the bonded product obtained in the above- mentioned step or a product resulting from the reaction in a soluble solvent and cleaving the tag from the bonded product or the resulting product (deprotection / cleavage step).

[0139] Step (1) (Dissolution Step)

[0140] In this step, the selectively soluble support compound of Formula (II) is dissolved in a soluble solvent.

[0141] As the solvent, a general organic solvent can be used. Since superior reactivity can be expected when the solubility in the solvent is higher, a solvent in which the compound of Formula (II) shows high solubility’ is preferably selected. Specifically, halogenated hydrocarbon such as chloroform, dichloromethane and the like; and a nonpolar organic solvent such as 1,4-dioxane, tetrahydrofuran and the like can be used. These solvents may be used in a mixture of two or more kinds thereof at an appropriate ratio. In addition, the above-mentioned halogenated hydrocarbons and nonpolar organic solvent may be mixed with aromatic hydrocarbons such as benzene, toluene, xylene and the like; nitriles such as acetonitrile, propionitrile and the like; ketones such as acetone, 2-butanone and the like; amides such as N,N-dimethylformamide and the like; sulfoxides such as dimethyl sulfoxide and the like at an appropriate ratio and used as long as the compound of the present invention is dissolved.

[0142] Step (2) (Bonding Step)

[0143] In this step, the compound of Formula (II) dissolved in a soluble solvent, which is obtained in the above-mentioned step (1) is bonded to a reaction substrate.

[0144] Here, the reaction substrate has a — COOH group of protected amino acid and the like, and the amount of the reaction substrate to be used is 1-10 mol, preferably 1-5 mol, per 1 mol of the compound of the present invention.(P101782W001)

[0145] When Y is a hydroxyl group, an ester bond can be formed by adding a condensing agent into a solvent that does not influence the reaction in the presence of a dimethylaminopyridine catalyst.

[0146] When Y is a group NHR. an amide bond can be formed by adding a condensing agent in the presence of a condensation additive (condensation promoter) such as 1- hydroxybenzotriazole (HOBt), ethyl l-hydroxy-lH-l,2,3-triazole-5-carboxylate (HOCt), 1- hydroxy-7-azabenzotriazole (HOAt) and the like.

[0147] The amount of the condensing additive to be used is preferably 0.05-1.5 mol per 1 mol of the compound of the present invention.

[0148] Examples of the condensing agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), N-ethyl-N'-3-dimethylaminopropylcarbodiimide and hydrochloride thereof (EDC HC1). hexafluorophosphoric acid (benzotriazol- 1- yloxy)tripyrrolizinophosphonium (PyBop), O-(benzotriazol-l-yl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TBTU), l-[bis(dimethylamino)methylene]-5-chloro- IH-benzotriazolium 3-oxide hexafluorophosphate (HCTU), O-benzotriazole-N,N,N'.N'- tetramethyluronium hexafluoroborate (HBTU), and the like.

[0149] The amount of the condensing agent to be used is 1-10 mol, preferably 1-5 mol. per 1 mol of the compound of the present invention.

[0150] Examples of the solvent include aromatic hydrocarbons such as toluene, xylene and the like; ethers such as diethyl ether, tetrahydrofuran, dioxane and the like; halogenated hydrocarbons such as chloroform, dichloromethane etc., and the like, and a mixture thereof. Of these, toluene, tetrahydrofuran and the like are preferable.

[0151] The reaction temperature is generally -10° C. to 30° C., preferably 0° C. to 20° C., and the reaction time is generally 1-30 hr.

[0152] For confirmation of the progress of the reaction, a method similar to general liquid phase organic synthesis reaction can be applied. That is, thin layer silica gel chromatography, high performance liquid chromatography and the like can be used to track the reaction.1HNMR may also be used to analyze / confirm the product, either in solution or as precipitated aliquot of the reaction mixture. The ability to use1HNMR is a benefit of using selectively soluble supports over solid-phase synthesis methods.(P101782W001)

[0153] Step (3) (Precipitation Step)

[0154] In this step, the bonded product obtained in the above-mentioned step (2), or a product obtained by dissolving the bonded product in a soluble solvent and performing a desired organic synthesis reaction is isolated by changing the solvent in which the bonded product or the resulting product is dissolved (e.g., change of solvent composition, change of solvent kind) to allow precipitation. That is, the reaction is performed under the conditions where the bonded product is dissolved and, after the reaction, the solvent composition is changed to allow precipitation of the bonded product through dilution of the reaction mixture with a polar solvent in which the product is insoluble. As the substitution solvent, polar organic solvents such as methanol, acetonitrile, and the like are used. That is, the reaction is performed under conditions where the compound can be dissolved and, for solvent substitution after the reaction, halogenated solvents, THF and the like are used for dissolution and polar organic solvents such as methanol, acetonitrile and the like are used for precipitation. Isolation by filtration and further washing of the isolated solid with additional portions of the polar solvent(s) should then afford the clean product.

[0155] Step (4) (Deprotection Step)

[0156] In this step, the selectively soluble support compound of Formula (II) is finally removed from the bonded product or resulting product isolated by precipitation in the above-mentioned step (3) to give the object compound.

[0157] When Y is a hydroxyl group, it reacts with — COOH of the initial reaction substrate to form an ester bond. After deprotection, the C-terminal of peptide becomes — COOH. On the other hand, when Y is an — NHR group, the — COOH group of the reaction substrate becomes an amide bond and, by removal of the support, the C-terminal is converted to a — CONHR group.

[0158] When Y is a hydroxyl group, the deprotection is preferably performed by an acid treatment. Examples of the acid to be used include trifluoroacetic acid (herein referred to as “TFA"’), hydrochloric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid and the like, with preference given to TFA. The deprotection is preferably performed under solution conditions using a solution such as chloroform, di chloromethane and THF, having an acid concentration of from about 0.1% to about 97%.(P101782W001)

[0159] The amount of the acid to be used is appropriately determined according to the kind of the acid to be used, and an amount suitable for removing the support is used. The amount that can be used is 3-100 mol. preferably 5-50 mol. per 1 mol of the bonded product. When an acid is used, trifluoromethanesulfonic acid, trimethylsilyl trifluoromethanesulfonate, BF3, Et20, and the like can also be added as a further strong acid source.

[0160] The reaction temperature is generally 0°C to 80° C, preferably 0°C to 30° C. The reaction time is generally 0.5-24 hr.

[0161] By utilizing the above-mentioned steps, a peptide can be produced. The compound of Formula (II) of the present invention can be mainly used as, but is not limited to, a protecting reagent for C-terminal of amino acid or peptide and the like. In addition, since the compound of Formula (II) wherein Y is a hydroxyl group can be converted to a corresponding chloroformate form by a method conventionally used in the pertinent field, for example, reaction with phosgene, the chloroformate form can also be used as a protecting reagent of N-terminal and the like.

[0162] A method of producing a peptide utilizing the above-mentioned steps, comprising the following steps;

[0163] (1) a step of obtaining C-protected amino acid or C-protected peptide, comprising condensing a compound of Formula (II) with the C-terminal of N-protected amino acid or N-protected peptide (C-terminal protection step),

[0164] (2) a step of removing a temporary protecting group of the N-terminal of the amino acid or peptide obtained in the above-mentioned step (deprotection step of N- terminal),

[0165] (3) a step of condensing the N-terminal of the amino acid or peptide obtained in the above-mentioned step with N-protected amino acid or N-protected peptide (peptide chain elongation step), and

[0166] (4) a step of precipitating the peptide obtained in the above-mentioned step (precipitation step).

[0167] Step (1) (C -Terminal Protection Step)(P101782W001)

[0168] In this step, the compound of Formula (II) is condensed with the C-terminal ofN- protected amino acid or N-protected peptide to give C-protected amino acid or C-protected peptide. For example, the step can be performed according to the above-mentioned binding step.

[0169] In the present invention, the “N-protected amino acid” and “N-protected peptide” mean amino acid and peptide wherein amino group is protected and carboxyl group is unprotected, and may be referred to as “P-AA-OH” (P is amino-protecting group (or temporary protecting group on demand)).

[0170] The condensation reaction of the compound of Formula (II) and C-terminal of N- protected amino acid or N-protected peptide is preferably performed in a solvent that does not influence the reaction. For example, the reaction is performed in the presence of a condensing agent when Y is a hydroxyl group or — NHR group, and an ester bond is formed when Y is hydroxyl, and amide bond is formed when Y is an — NHR group. Examples of the condensing agent include di cyclohexylcarbodiimide. diisopropylcarbodiimide, N-ethyl-N'-3-dimethylaminopropylcarbodiimide and a hydrochloride thereof (EDC.HC1), and the like. An ester bond forming reaction is performed in the presence of dimethylaminopyridine, and an amide bond forming reaction is performed in the presence of a condensation additive such as HOBt, HOCt and the like.

[0171] Examples of the solvent to be used for the step include halogenated hydrocarbons such as chloroform, dichloromethane and the like; and nonpolar organic solvents such as 1,4-di oxane, tetrahydrofuran and the like. These solvents may be used in a mixture of two or more kinds thereof at an appropriate ratio, and preferred is dichloromethane. The amount of the solvent to be used is preferably 2- to 50-fold volume relative to the benz lic compound of the present invention.

[0172] The reaction temperature is generally -10 °C to 40 °C. preferably 0 °C to 30 °C. The reaction time is generally 1-70 hr.

[0173] Step (2) (N-Terminal Deprotection Step)

[0174] In this step, the temporary protecting group of the N-terminal of amino acid or peptide obtained in the above-mentioned step (1) is removed.(P101782W001)

[0175] As the temporary protecting group of the N-terminal, the below-mentioned aminoprotecting groups generally used in the technical field of peptide chemistry and the like are usable. In the present invention, a tert-butoxy carbonyl group (hereinafter to be also referred to as Boc group), or a 9-fluorenylmethoxy carbonyl group (hereinafter to be also referred to as Fmoc group) are / is preferably used.

[0176] While the deprotection conditions are appropriately selected depending on the kind of the temporary' protecting group, a group that can be removed under conditions different from removal of the protecting reagent derived from the compound of the present invention is preferable. For example, Fmoc group can be removed by a treatment with a base, and Boc group can be removed by a treatment with an acid. The reaction is performed in a solvent which does not influence the reaction.

[0177] Examples of the base include dimethylamine, diethylamine, piperidine, and the like. 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 diethyl ether, tetrahydrofuran, dioxane, and the like; nitriles such as acetonitrile and the like, and a mixture thereof.

[0178] Step (3) (Peptide Chain Elongation Step)

[0179] In this step, the N-terminal of amino acid or peptide deprotected in N-terminal in step (2) is condensed with N-protected amino acid or N-protected peptide.

[0180] This step is performed by using the condensing agent, condensation additive and the like described in the aforementioned step (1) and under peptide synthesis conditions generally used in the field of peptide chemistry.

[0181] Step (4) (Precipitation Step)

[0182] This step is performed in the same manner as in the precipitation step in the above-mentioned step (3).

[0183] In the production method of the peptide of the present invention, the N-protected amino acid or N-protected peptide obtained in step (4) can be subjected to steps (5)-(7) in a desired number of repeats:

[0184] (5) a step of removing the temporary protecting group of N-terminal of the peptide obtained in the precipitation step,(P101782W001)

[0185] (6) a step of condensing N-protected amino acid or N-protected peptide with N- terminal of the peptide obtained in the above-mentioned step, and

[0186] (7) a step of precipitating the peptide obtained in the above-mentioned step.

[0187] Step (5)

[0188] The step is performed in the same manner as in the deprotection step of N- terminal in step (2).

[0189] Step (6)

[0190] This step is performed in the same manner as in the peptide chain elongation step of step (3).

[0191] Step (7)

[0192] This step is performed in the same manner as in the precipitation step of step (3).

[0193] In the production method of the peptide of the present invention, a step of deprotecting the C-terminal of the peptide, wherein the C-terminal is protected with a compound of Formula (II). may be further contained after the precipitation step of step (4) or step (7). For example, the step can be performed according to the above-mentioned step (4) for deprotection of the anchor of the present invention.

[0194] The resultant peptide as the final target can be isolated and purified according to methods normally used in peptide chemistry. For example, the reaction mixture can be subjected to extraction washing, cry stallization, chromatography and the like, to isolate and purify a peptide as the final target.

[0195] When the organic synthesis reaction or peptide synthesis reaction of the present invention contains multi-steps, the aforementioned precipitation step may be appropriately omitted as long as the reaction in the next step is not influenced.

[0196] In each reaction, when the starting compound has a hydroxy group, an amino group, a carboxy group or a carbonyl group (particularly when amino acid or peptide has a functional group in the side chain), a protecting group generally used in the peptide chemistry' and the like may be introduced into these groups, and the object compound can be obtained by removing the protecting group as necessary after the reaction.(P101782W001)

[0197] In a final step (excluding any purification steps), the peptide is cleaved 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.

[0198] Specific examples of liquid phase peptide production with partially soluble supports can be found in, for example, U.S. Patent No. 11,420,997, U.S. Publication No. 2019 / 0023726, U.S. Publication No. 2011 / 0160433, U.S. Publication No. 2018 / 0215782, and U.S. Patent No. 9,206,230, the entireties of which are incorporated herein by reference.

[0199] Composition Changing Means

[0200] As the means for changing the composition of a solution which is preferably used in 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 wi th 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 which is selected to solubilize the impurities over the precipitated tag-protected supported oligonucleotide

[0201] The lower solubility antisolvent solvent used in the present invention means a solvent 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 alkyd, alcohols and any proportioned aqueous methanol or lower alkyl alcohols (z.e., C1-C4) and water.(P101782W001)

[0202] This procedure using the selectively soluble support compounds of Formula (II) can also be used to synthesize an oligonucleotide in a liquid phase synthesis.

[0203] One skilled in the art, using the preceding detailed description, can utilize the present 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.EXAMPLESExample 1: Synthesis of Tag 5 (Nl.N3-dioctadecyl-5-((6-oxo-6-(piperazin-l-yl)- hexylloxylisophthal amide)5-acetoxyisophthalic acid (2):(P101782W001)

[0204] 5-hydroxyisophthalic acid 1 (10.0 g, 54.9 mMol, 1.0 eq) was added in to a flask containing pyridine (20 mL, 2.0v) and cooled to 10-15 °C. To the suspension, acetic anhydride was added slowly over 15-30 minutes at 10-20 °C. Aliquots were monitored by TLC for reaction completion. After 40 hours, all 1 was consumed and the reaction mixture was added into 2 AHC1 solution (lOOmL, lOv) at 0-10 °C. Extracted the product in ethyl acetate and precipitated with heptane to afford 5-acetoxyisophthalic acid 2 as white solid (12.5g, Quantitative). FIG. 1 shows theXH NMR (600 MHz, CD3OD) of compound 2.1H NMR (CD3OD, 600 MHz) 5 8.55 (1H), 7.98 (2H), 2.38 (3H).3.5-bis(octadecylcarbamoyl)phenyl acetate (3):

[0205] 5-acetoxyisophthalic acid 2 (3.0g, 13.38mMol. 1.0 eq) was added in to a flask containing DCM (60 mL, 20.0v). To the suspension, stearyl amine (7.93g, 29.44mMol, 2.2 eq.) was added and cooled to 10-15 °C. To the white suspension, T3P solution (50% soln., in ACN, 25.6g, 40.15mMol, 3.0 eq.) was added slowly over 15-30 minutes followed by DIPEA (5.19g, 40.15mMol, 3.0 eq.). Monitored the reaction by 'H NMR for reaction completion. After Ih, all 2 was consumed and the solid was filtered to afford 3,5- bis(octadecylcarbamoyl)phenyl acetate 3 as white solid (9.1g, 93.5%). FIG. 2 shows the 'H NMR (600 MHz, CDCI3) of compound 3.JH NMR (CDCI3, 600 MHz) 5 8.05 (IH), 7.65 (2H), 6.41 (2H), 3.40-3.46 (4H), 2.35 (3H), 1.60-1.68 (4H), 1.20-1.50 (60H), 0.85- 0.92 (6H).5-hydroxy-Al ,A3-dioctadecylisophthalamide (4):(P101782W001)

[0206] 3,5-bis(octadecylcarbamoyl)phenyl acetate 3 (9.0g, 12.38mMol, 1.0 eq) was added in to a flask containing methanol-THF mixture (135mL, 15. Ov, 2: 1). To the suspension, 20% LiOH solution (0.44g, 18.57mMol, 1.5 eq.) was added and raised the temperature to 50-55 °C. Monitored the reaction by 'H NMR for reaction completion. After 6h. all 3 was consumed and concentrated the reaction mixture. Added methanol to the crude (90mL, lOv) and filtered to afford 5 -hydroxy- AH ,A3-dioctadecylisophthal amide 4 as white solid (8.0g, 94.4%). FIG. 3 shows the!H NMR (600 MHz, CDCh) of compound 4. 'H NMR (CDCh, 600 MHz) 5 7.85 (1H), 7.42 (2H), 7.18 (2H), 3.42-3.48 (4H), 1.60- 1.68 (4H), 1.12-1.45 (60H), 0.82-0.90 (6H). / -butyl 4-(6-(3.5-bis(octadecylcarbamoyl)phenoxy)hexanoyl)piperazine-l -carboxylate (8):

[0207] 5-hydroxy-Afl ,N3-dioctadecylisophthalamide 4 (2.0g, 2.92mMol, 1.0 eq) was added in to a flask containing 2-methyl THF (20mL, 10. Ov). To the suspension, tert-butyl 4-(6-bromohexanoyl)piperazine-l-carboxylate 7 (1.38g, 3.79mMol, 1.3 eq.) followed by potassium carbonate (1.01g, 7.30mMol, 2.5 eq.) was added and raised the temperature to 80-85 °C. Monitored the reaction by 'H NMR for reaction completion after 48h.Concentrated the reaction mixture and added water (20mL, lOv). Extracted the product in DCM and precipitated with acetonitrile to afford / -butyl 4-(6-(3,5- bis(octadecylcarbamoyl)phenoxy)hexanoyl)piperazine-l -carboxylate 8 as white solid (2.5g, 88.7%). FIG. 4 shows the 'H NMR (600 MHz. CDCI3) of compound 8. 'H NMR (CDCls, 600 MHz) 5 7.71 (1H), 7.45 (2H), 6.30 (2H), 4.05-4.10 (2H), 3.60-3.65 (2H), 3.40-3.50 (10H), 2.38-2.42 (2H), 1.82-1.86 (2H), 1.72-1.76 (2H), 1.18-1.68 (75H), 0.88- 0.92 (6H).(P101782W001)7V1.7V3-dioctadecyl-5-((6-oxo-6-(piperazin-l-yl)hexyl)oxy)isophthalamide (9):

[0208] / -butyl 4-(6-(3,5-bis(octadecylcarbamoyl)phenoxy)hexanoyl)piperazine- 1 - carboxylate 8 (2.5g, 2.58mMol, 1.0 eq) was added in to a flask containing DCM (25mL, 10. Ov). To the clear solution, trifluoroacetic acid (2.5mL. l.Ov) was added and continued to stir at 25-30 °C. Monitored the reaction by TLC for reaction completion after 22h. Concentrated the reaction mixture, then neutralized with sodium bicarbonate solution and extracted the product in DCM and precipitated with acetonitrile to afford N1,N3- dioctadecyl-5-((6-oxo-6-(piperazin-l-yl)hexyl)oxy)isophthalamide 9 as white solid (2.19g, 97.3%). FIG. 5 shows the ' H NMR (600 MHz, CDCh) of compound 8. ' H NMR (CDCh, 600 MHz) 5 7.71 (1H), 7.45 (2H), 6.41 (2H), 4.05-4.10 (2H), 3.60-3.65 (2H), 3.40-3.50 (6H), 2.85-2.92 (4H), 2.35-2.42 (2H), 1.82-1.86 (2H), 1.72-1.76 (2H), 1.20-1.70 (67H), 0.85-0.92 (6H). / -butyl 4-(6-bromohexanoyl)piperazine-l -carboxylate (7):

[0209] 6-bromohexanoic acid 5 (5.0g, 25.6mMol, 1.0 eq) was added in to a flask containing DCM (lOOmL, 20.0v). To the clear solution, / -buty l piperazine- 1 -carboxylate 6 (5.3g, 28.2mMol, 1.1 eq.) followed by EDC.HC1 (5.9g, 30.8mMol, 1.2 eq.) was added and continued to stir at 25-30 °C. Monitored the reaction by TLC for reaction completion after 17h. Washed the reaction mixture with sodium bicarbonate solution and 17V HO solution and precipitated with heptane to afford / -butyl 4-(6-bromoh exanoy l)piperazine-l- carboxylate 7 as white solid (7.6g, 81.7%). FIG. 6 shows the ’H NMR (600 MHz, CDCh) of compound 7. 'H NMR (CDCh, 600 MHz) 5 3.60-3.65 (2H), 3.40-3.50 (8H), 2.35-2.40 (2H), 1.90-1.95 (2H), 1.65-1.72 (2H), 1.45-1.55 (11H).Example 2: Oligonucleotide Tetramer Synthesis with Selectively Soluble Compound 9(P101782W001)

[0210] The following schemes show a linear and a convergent oligonucleotide synthesis using selectively soluble compound 9:Linear:Convergent:(P101782W001)

[0211] Al,A3-dioctadecyl-5-((6-oxo-6-(piperazin-l-yl)hexyl)oxy)isophthalamide (TAG) 9 (2.0g, 2.31mMol, 1.0 eq) was added in to a flask containing DCM (40mL, 20.0v). To the clear solution, DMTr-5’-dG(iBu)-3’-succinate 10 (1.88g, 2.54mMol, 1.1 eq.) followed by DIPEA (0.45g. 3.46mMol. 1.5 eq.) was added and continued to stir at 25-30 °C. To the pale-yellow clear solution, T3P solution (50% in acetonitrile, 2.0mL, 3.46mMol, 1.5 eq.) was added slowly in 10-15 minutes and continued to stir at 25-30 °C. Monitored the reaction by TLC after Ih. Washed the reaction mixture with sodium bicarbonate solution and water. Concentrated the organic layer and precipitated with acetonitrile. The crude solid was treated with dichloroacetic acid in DCM to cleave DMTr group and precipitated with acetonitrile to afford TAG-3 ’dG(iBu)-5’ -OH 11 as white solid (2.1g, 70.7%). FIG. 7 shows the 'H NMR (600 MHz, CDC13) of compound 11.JH NMR (CDCI3, 600 MHz) 5 7.85 (IH), 7.63 (IH), 7.25-7.45 (4H), 6.15-6.20 (IH), 5.42-5.46 (IH), 4.20 (IH), 3.95-4.05 (2H). 3.80-3.86 (2H), 3.00-3.75 (19H), 2.90-2.98 (IH), 2.60-2.70 (5H). 2.40-2.45 (IH), 2.30-2.38 (2H), 1.40-1.90 (10H), 1.05-1.40 (60H), 0.82-0.88 (6H).General Procedure A:(P101782W001)

[0212] TAG-succinate-3'-Oligo-5'-OH (1.0 eq) was added in to a flask containing DCM (lO.Ov). To the clear solution, DMTr-Oligo-amidite (1.1 eq.) followed by molecular sieves 3 A (1.0T) was added and continued to stir at 25-30 °C. To the suspension, 4,5- dicyanoimidazole (1.5 eq.) was added and continued to stir at 25-30 °C for Ih. After completion of the reaction, Xanthane hydride (1.1 eq.) was added and continued to stir for Ih. Monitored the completion of the reaction by 3 IP NMR. To the suspension, 1- dodecanethiol (3.0eq.) followed by trifluoroacetic acid (7.0 eq.) was added and continued to stir for 2h. Quenched the reaction mixture with N-methyl imidazole. Filtered the molecular sieves and washed with sodium bicarbonate solution and water. Concentrated the DCM layer and precipitated with acetonitrile to afford TAG-succinate-3’Oligo-5’OH as white solid (80-95%).TAG-3'-dG(iBu)-dC(Bz)-5'-OH (12):

[0213] Following General Procedure A, TAG-3 ’-dG(iBu)-dC(Bz)-5’ -OH was isolated as white solid (97.4%). FIG. 8 shows the 'H NMR (600 MHz, CDCh) of compound 12.1H NMR (CDCh, 600 MHz) 5 8.35-8.38 (1H), 7.88-8.95 (3H), 7.30-7.70 (7H), 6.18-6.28 (2H), 5.90 (1H), 5.45-5.51 (1H), 4.98-5.20 (1H), 4.15-4.40 (5H), 3.95-4.05 (2H), 3.30-3.85 (17H), 2.50-3.20(22H), 2.30-2.40 (3H), 1.40-1.85 (11H), 1.00-1.40 (56H), 0.80-0.90 (6H).31P (CDCh, 600Hz) 566.07, 66.48. FIG. 9 is a31P NMR (600 MHz, CDCh) of compound 12.TAG-3'-dG(iBu)-dC(Bz)-dG(iBu)-5'-OH (13):(P101782W001)

[0214] Following General Procedure A, TAG-3'-dG(iBu)-dC(Bz)-dG(iBu)-5’-OH (13) was isolated as white solid (75%). FIG. 10 shows the 'H NMR (600 MHz, CDCh) of compound 13. 'H NMR (CDCh, 600 MHz) 5 8.05-8.20 (1H), 7.80-8.00 (4H), 7.30-7.80 (7H), 7.15-7.22 (2H), 6.10-6.30 (3H), 5.05-5.50 (4H), 4.15-4.50 (11H), 3.95-4.05 (2H), 3.30-3.85 (14H), 2.50-3.20 (22H), 2.30-2.40 (3H), 1.40-1.85 (10H), 1.00-1.40 (70H), 0.80- 0.90 (6H). FIG. 11 is a31P NMR (600 MHz, CDCh) 566.51 of compound 13.

[0215] 50 mg of the solid 13 was treated with NH4OH (0.5 mL, 28-30%) and the mixture was stirred at 65 °C for 4 h. An aliquot was diluted with MeCN / H2O (1 : 1) and the mixture was filtered (0.2 pm filter). The filtrate was analyzed by LCMS. MS (ESI+): m+z / z calcd for OH-3’-GCG-5’-OH [C29H37N13O14P2S2 + H]+: 918.1, found: 918.1.Synthesis of Fragment DMTr-5’-CTT-3?-Phosphoramidite(P101782W001)15 16DMTr-5'-dT-3'OH 5'-OH-dT-3'-TBDPS

[0216] DMTr-5’-dT-3’-OH 15 (3.0g, 5.51mMol, 1.0 eq) was added in to a flask containing DCM (30mL, 10. Ov). To the clear solution, TBDPS chloride (2.27g, 8.26mMol, 1.5 eq.) followed by Imidazole (1. 13g. 16.53mMol, 3.0 eq.) was added and continued to stir at 25-30 °C. Monitored the reaction by TLC after 12h and quenched the excess TBDPS chloride with 2 -propanol. To the pale-yellow solution, 1 -dodecanethiol (1.72mL, 7.16mMol, 1.3 eq.) and dichloroacetic acid (2.27mL, 27.54mMol, 5.0 eq.) was added and(P101782W001) continued to stir for 2h at 25-30 °C. Washed the reaction mixture with sodium bicarbonate solution and precipitated with MTBE to afford TBDPS-3'-dT-5'-OH 16 as white solid (2.6g, 98%). FIG. 12 shows the 'H NMR (600 MHz, CDCh) of compound 16. 'H NMR (CDCh, 600 MHz) 5 8.19 (1H), 7.65-7.72 (4H), 7.40-7.50 (6H), 7.28-7.32 (1H), 6.23-6.28 (1H), 4.47-4.50 (1H), 4.00 (1H), 3.65-3.70 (1H), 3.25-3.30 (1H), 2.25-2.35 (1H), 2.15-2.22 (1H), 1.95-2.00 (1H), 1.89 (3H), 1.11 (9H).5’-OH-dT-dT-3’-TBDPS (17):

[0217] DMTr-5’-dT-3’-TBDPS 16 (3.0g, 6.24mMol, 1.0 eq) was added in to a flask containing DCM+ACN (1: 1. 30mL, 10. Ov). To the clear solution, DMTr-dT-amidite (5.11 g. 6.87mMol, 1.1 eq.) followed by molecular sieves 3 A (3.0g, 1.0T) was added and continued to stir at 25-30 °C. To the suspension, 4,5-dicyanoimidazole (1.11g, 9.36mMol,1.5 eq.) was added and continued to stir at 25-30 °C for Ih. After completion of the reaction, Xanthane hydride (1.03g. 6.87mMol. 1.1 eq.) was added and continued to stir for Ih. Monitored the completion of the reaction by31P NMR. To the suspension. 1- dodecanethiol (4.49mL, 18.73mMol, 3.0eq.) followed by trifluoroacetic acid (3.35mL, 43.69mMol, 7.0 eq.) was added and continued to stir for 2h. Quenched the reaction mixture with N-methyl imidazole. Filtered the molecular sieves and washed with sodium bicarbonate solution and water. Concentrated the DCM layer and precipitated with MTBE to afford 5’-OH-dT-dT-3’TBDPS as white solid (4.92g, 92.3%). FIG. 13 shows the 'H NMR (600 MHz, CDCh) of compound 17. 'H NMR (CDCI3, 600 MHz) 5. FIG. 14 shows the31P NMR (600 MHz, CDCh) of compound 17.5’-OH-dT-dT-3’-dC(Bz)-3’-QH (18):(P101782W001)

[0218] 5’-OH-dT-dT-3 -TBDPS 17 (4.90g, 5.74mMol, 1.0 eq) was added in to a flask containing DCM+ACN (1 :1 , 49mL, 10.Ov). To the clear solution, DMTr-dC(Bz)-amidite (5.35g, 6.31mMol, 1.1 eq.) followed by molecular sieves 3 A (4.9g, 1.0T) was added and continued to stir at 25-30 °C. To the suspension, 4,5 -di cyanoimidazole (1.02g, 8.61mMol, 1.5 eq.) was added and continued to stir at 25-30 °C for Ih. After completion of the reaction, Xanthane hydride (0.95g, 6.31mMol, 1.1 eq.) was added and continued to stir for Ih. Monitored the completion of the reaction by31P NMR. To the suspension, a solution of imidazole (11.72g, 172. 14mMol, 30.0eq.) and pyridine.HF (2.2mL, 86.07mMol, 15.0 eq.) in THF (49mL, lO.Ov) was added at 0-5 °C. Continued to stir for 2h at 0-5 °C and monitored by HPLC. Filtered the molecular sieves and washed with sodium bicarbonate solution and water. Concentrated the DCM layer and precipitated with heptane to afford 3’-OH-dT-dT-dC(Bz)-5’DMTr as white solid (5.30g, 57.6%). FIG. 15 shows the 'H NMR (600 MHz, CDCh) of compound 18. ' H NMR (CDCh, 600 MHz) 5.DMTr-5’dC(Bz)-dT-dT-3'-phosphoramidite (19):(P101782W001)

[0219] DMTr-5’-dC(Bz)-dT-dT-3'-OH 17 (5.0g, 3.66mMol, 1.0 eq) was added in to a flask containing THF (50mL, lO.Ov). To the cooled solution, 2-cyanoethyl tetrapropylphosphorodiamidite (3.31g. 10.98mMoL 3.0 eq.) was added followed by pyridine trifluoroacetate (1.42g, 7.33mMol, 2.0 eq.) and continued to stir at 0-5 °C for Ih. After completion of the reaction, Xanthane hydride (0.95g, 6.31mMol, 1.1 eq.) was added and continued to stir for Ih. Precipitated the product with MTBE and heptane mixture to afford DMTr-5?-dC(Bz)-dT-dT-3’-phosphoramidite 19 as a white solid (5.04g. 88%). 'H NMR (CDCh, 600 MHz) 5. FIG. 16 shows the1H NMR (600 MHz, CDCh) of compound18Synthesis of Fragment TAG-3 GCGTTCTTCTTC-5 ’-OH(P101782W001)

[0220] TAG-3 ’dG(iBu)-dC(Bz)-dG(iBu)-5’ -OH 13 (700mg, 0.32mMol, 1.0 eq) was added in to a flask containing DCM (7mL, lO.Ov). To the clear solution, DMTr-5’-dC(Bz)- dT-dT-3’-phosphoramidite (649mg, 0.41mMol, 1.3 eq.) followed by molecular sieves 3 A (700mg, 1.0T) was added and continued to stir at 25-30 °C. To the suspension, 4,5- dicyanoimidazole (56mg, 0.47mMol, 1.5 eq.) was added and continued to stir at 25-30 °C for Ih. After completion of the reaction, Xanthane hydride (52mg, 0.35mMol, 1.1 eq.) was added and continued to stir for Ih. Monitored the completion of the reaction by31P NMR. To the suspension, ethanol (145mg, 3.16mMol, lO.Oeq.) followed by trifluoroacetic acid (180mg, 1.58mMol, 5.0 eq.) was added and continued to stir for 2h. Quenched the reaction mixture with N-methyl imidazole. Filtered the molecular sieves and washed with sodium bicarbonate solution and water. Concentrated the DCM layer and precipitated with acetonitrile to afford TAG-3 ’dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-dC(Bz)-5 ’-OH 20 as a white(P101782W001) solid (610mg, 56.5%). FIG. 17 shows theJH NMR (CDCh, 600 MHz) 5 of compound 20. FIG. 18 is the31P NMR (600 MHz, CDCh) of compound 20.

[0221] 25 mg of the solid 20 was treated with NH4OH (0.5 mL, 28-30%) and the mixture was stirred at 65 °C for 4 h. An aliquot was diluted with MeCN / H2O (1 : 1) and the mixture was filtered (0.2 pm filter). The filtrate was analyzed by LCMS. MS (ESI+): m+z / z calcd for OH-3’-GCGTTC-5’-OH [C58H75N20O31P5S5 + 2H]+ / 2: 932.1, found: 932.3.TAG-3’-dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-dC(Bz)-dT-dT-dC(Bz)-5’-OH (21):

[0222] TAG-3'dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-dC(Bz)-5’-OH 20 (500mg, 0.15mMol, 1.0 eq) was added in to a flask containing DCM (lOmL, 20. Ov). To the clear solution, DMTr-5’-dC(Bz)-dT-dT-3’-phosphoramidite (254mg, 0.16mMol, 1.1 eq.) followed by molecular sieves 3 A (500mg, 1.0T) was added and continued to stir at 25-30 °C. To the suspension, 4, 5-dicy anoimidazole (26mg, 0.22mMol, 1.5 eq.) was added and continued to stir at 25-30 °C for Ih. After completion of the reaction, Xanthane hydride (24mg. 0.16mMol, 1.1 eq.) was added and continued to stir for Ih. Monitored the completion of the reaction by31P NMR. To the suspension, ethanol (67mg, 1.46mMol, lO.Oeq.) followed by trifluoroacetic acid (83mg, 0.73mMol, 5.0 eq.) was added and continued to stir for 2h. Quenched the reaction mixture with N-methyl imidazole. Filtered the molecular sieves and washed with sodium bicarbonate solution and water. Concentrated the DCM layer and precipitated with acetonitrile to afford TAG-3 ’ dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-dC(Bz)- dT-dT-dC(Bz)-5'-OH 21 as a white solid (550mg, 81.4%). FIG. 19 shows the 'H NMR (CDCh, 600 MHz) 5 of compound 21. FIG. 20 shows the31P NMR (600 MHz, CDCh) of compound 21.

[0223] 25 mg of the solid 21 was treated with NH4OH (0.5 mL. 28-30%) and the mixture was stirred at 65 °C for 4 h. An aliquot was diluted with MeCN / H2O (1 : 1) and the mixture(P101782W001) was filtered (0.2 pm filter). The filtrate was analyzed by LCMS. MS (ESI+): m+z / z calcd for OH-3’-GCGTTCTTC-5'-OH [C87H113N27O48P8S8 + 3H]+ / 3: 936.8, found: 937.4.TAG-3’-dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-dC(Bz)-dT-dT-dC(Bz)-dT-dT-dC(Bz)-5’-OH(22):

[0224] TAG-3’dG(iBu)-dC(Bz)-dG(iBu)-dT-dT-dC(Bz)-dT-dT-dC(Bz)-5’-OH 21 (500mg, O.llmMol, 1.0 eq) was added in to a flask containing DCM (lOrnL, 20. Ov). To the clear solution, DMTr-5’-dC(Bz)-dT-dT-3’-phosphoramidite (222mg, 0.16mMol, 1.3 eq.) followed by molecular sieves 3 A (500mg, 1.0T) was added and continued to stir at 25-30 °C. To the suspension, 4,5-dicyanoimidazole (19mg. 0. 16mMol, 1.5 eq.) was added and continued to stir at 25-30 °C for Ih. After completion of the reaction, Xanthane hydride (18mg, 0. 12mMol, 1. 1 eq.) was added and continued to stir for Ih. Monitored the completion of the reaction by31P NMR. To the suspension, ethanol (50mg, 1.08mMol, lO.Oeq.) followed by trifluoroacetic acid (61mg. 0.54mMol. 5.0 eq.) was added and continued to stir for 2h. Quenched the reaction mixture with N-methyl imidazole. Filtered the molecular sieves and washed with sodium bicarbonate solution and water.Concentrated the DCM layer and precipitated with acetonitrile to afford TAG-3 ’dG(iBu)- dC(Bz)-dG(iBu)-dT-dT-dC(Bz)-dT-dT-dC(Bz)-dT-dT-dC(Bz)-5'-OH 22 as a white solid (340mg, 54%). FIG. 21 shows the 'H NMR (CDCh, 600 MHz) 6 of compound 22. FIG. 22 shows the31P NMR (600 MHz, CDCI3) of compound 22.

[0225] 25 mg of the solid 22 was treated with NH4OH (0.5 mL, 28-30%) and the mixture was stirred at 65 °C for 4 h. An aliquot was diluted with MeCN / H2O (1 : 1) and the mixture was filtered (0.2 pm filter). The filtrate was analyzed by LCMS. MS (ESI+): m+z / z calcd for OH-3’-GCGTTCTTCTTC-5’-OH [C116H151N34O65P11S11 + 3H]+ / 3: 1251.8, found: 1252.3.Example 3; Synthesis of Tag 5P-S(P101782W001)4-(3-Bromopropoxy)-2-methoxybenzaldehyde (25):

[0226] Benzaldehyde 23 (5.00 g. 32.9 mmol, 1.0 eq) was added to a flask followed by acetonitrile (10 Vol, 50 mL) and stirred at ambient temperature under anitrogen atmosphere, forming a slurry. Next, potassium carbonate (13.6 g, 98.6 mmol, 3.0 eq) and 1,3-di bromopropane (66.3 g, 329 mmol, 10 eq) were added and the reaction mixture was heated to 55 °C, resulting in a pale brown slurry. After stirring for 4 hours, the reaction was removed from heat, cooled, and filtered. The wet cake was washed once with acetonitrile (20 mL). The mother liquor was reduced to an oil by rotary evaporation and loaded onto a pre-equilibrated 220 g SiCh column and eluted in a gradient of 0-50% EtOAc in heptane. Fractions containing the desired product w ere combined and evaporated to produce a w et solid. The wet solid was transferred to a vacuum oven and dried for 18 hours at 40 °C to afford 6.70 g of 25 as a white solid (75% yield). FIG. 23 shows the 1H NMR (400 MHz. CDC13) of compound 25. 'H NMR (400 MHz, CDCh) 5 10.29 (d, J= 0.8 Hz, 1H), 7.80 (d, J= 8.7 Hz, 1H), 6.55 (ddd, J= 8.8, 2.2, 0.8 Hz, 1H), 6.46 (d, J= 2.2 Hz, 1H), 4.19 (t, J = 5.8 Hz, 2H), 3.90 (s, 3H), 3.60 (t, J= 6.3 Hz, 2H), 2.34 (p, J= 6.1 Hz, 2H).5-(3-(4-Formyl-3-methoxyphenoxy)propoxy)- dioctadecylisophthal amide (26):(P101782W001)

[0227] Phenol 4 (from Example 1) (4.0 g, 5.8 mmol, 1.0 eq) was added to a flask followed by 2-Me THF (6.3 Vol, 25 mL) and stirred at ambient temperature under a nitrogen atmosphere, forming a slurry. Next, potassium carbonate (3.2 g, 23 mmol. 4.0 eq), potassium iodide (1.3 g, 7.6 mmol, 1.3 eq), and aldehyde 25 (2.1 g. 7.6 mmol, 1.3 eq) were added and the reaction mixture was heated to reflux. After refluxing for 24 hours, the reaction mixture was cooled to room temperature then the solvent was removed to yield a solid. Water (40 Vol. 10 mL) and DCM (100 mL) were added, and the resulting biphasic mixture was stirred for -15 minutes until all suspended solids dissolved. The biphasic mixture was transferred to a separatory funnel and the organic layer was drained. The aqueous layer was washed with DCM (2 x 100 mL) then the combined organic layers were reduced to -10 mL via rotary evaporation. Acetonitrile (100 mL) was added slowly to the remaining 10 mL and the resulting slurry’ was agitated for 5 hours before it was filtered. The isolated solid was washed with acetonitnle (100 mL) then transferred to a vacuum oven and dried at 40 °C overnight. After drying for -18 hours, a total of 4.4 g of 26 was isolated as an off-white solid (86% yield). FIG. 24 shows the 1H NMR (400 MHz, CDC13) of compound 26. 'H NMR (400 MHz, CDC13) 8 10.28 (d, J= 0.7 Hz, 1H), 7.79 (d, J= 8.6 Hz. 1H), 7.67 (t, J = 1.5 Hz, 1H), 7.45 (d, J= 1.4 Hz, 2H), 6.59 - 6.42 (m, 2H), 6.24 (d, J = 5.7 Hz, 2H), 4.24 (dt, J= 11.9, 6.0 Hz, 4H), 3.90 (s, 3H), 3.43 (q, J= 6.7 Hz, 4H), 2.30 (p, J= 6.0 Hz, 2H), 2.00 (s, 26H), 1.61 (dd, J= 12.4, 6.0 Hz, 8H), 1.25 (s, 59H), 0.92 - 0.78 (m, 7H). FIG. 25 shows the LCMS (ES+) chromatograph of compound 26. LCMS m / z found: 877.7028 (M+H+).5-(3-(4-(Hvdroxymethyl)-3-methoxyphenoxy)propoxy) dioctadecylisophthal amide(27, Tag 5P-S):Tag 5P-S

[0228] Aldehyde 26 (4.2 g, 4.8 mmol, 1.0 eq) was added to a flask followed by DCM (10 Vol, 42 mL) and stirred at ambient temperature under a nitrogen atmosphere. After stirring(P101782W001) for 10 minutes, all the starting material dissolved MeOH (10 Vol, 42 mL) was added to the flask. Sodium borohydride (0.36 g, 9.6 mmol, 2.0 eq) was added and the reaction was allowed to continue stirring at ambient temperature. After stirring for 90 minutes, the reaction was quenched with water (45 mL) followed by 1 M HO (45 mL) and stirred for 15 minutes. The biphasic organic layer was removed on a rotary evaporator, forming a precipitate in the remaining aqueous liquid. The flask was cooled in an ice bath and agitated for 30 minutes at 5-10 °C. The slurry was then filtered and the wet cake was washed with water (3 x 20 mL). The isolated solid was transferred to a vacuum oven and dried at 40 °C overnight. After dry ing for 2 days, a total of 3.9 g of 27 was isolated as an off-white solid (92% yield). FIG. 26 shows the 'l l NMR (400 MHz, CDC13) of compound 27. 'H NMR (400 MHz, CDCh) 5 7.66 (t, J= 1.6 Hz, 1H), 7.43 (d, J= 1.5 Hz, 2H), 7.14 (d, J= 8.1 Hz, 1H), 6.49 - 6.40 (m, 2H), 6.24 (t, J= 5.8 Hz, 2H), 4.56 (d, J= 33.5 Hz, 2H), 4.25 (t, J = 6.0 Hz, 2H), 4. 15 (t, J = 6.0 Hz, 2H), 3.81 (d, J = 19.4 Hz, 3H), 3.43 (td, J = 7.2, 5.6 Hz, 4H), 2.27 (p, J= 6.1 Hz, 2H), 1.59 (q, J= 7.0 Hz, 5H), 1.25 (s, 62H), 0.91 - 0.83 (m, 6H).Example 4; Peptide Hexamer Synthesis with Selectively Soluble Compound 27Fmoc-Phe-Gly-Gly-Tae5P-S (29):(P101782W001)

[0229] Tag 5P-S (27) (1.50 g, 1.71 mmol, 1.0 eq) was added to a flask followed by DCM (40 Vol, 60 mL) and stirred at ambient temperature under a nitrogen atmosphere, forming a clear solution. Next, EDCI’HCl (0.49 g, 2.56 mmol. 1.5 eq) was added followed by DMAP (0.042 g, 0.341 mmol, 0.2 eq) and Fmoc-PheGlyGly-OH (1.28 g, 2.56 mmol. 1.5 eq). The resulting slurry was allowed to stir overnight at ambient temperature. After stirring for 24 hours, the reaction mixture was concentrated to dryness on a rotary evaporator. Acetone (150 mL) was added and the resulting slurry was stirred for 2 hours before it was filtered. The filter cake was rinsed with acetone (50 mL) and the gel-like solid was transferred to a vacuum oven and dried overnight under vacuum at ambient temperature. After drying for ~18 hours, 2.01 g of 29 was isolated as an off-white solid (87% yield). FIG. 27 shows the1H NMR (400 MHz, CDC13) of compound 29. FIG. 28 shows the LCMS (ES+) chromatograph of compound 29. LCMS m / z found: 1362.8979 (M+H+).H-Phe-Gly-Gly-Tag5P-S (30):

[0230] Fmoc-Phe-Gly-Gly-Tag5P-S 29 (1.90 g, 1.4 mmol, 1.0 eq) was added to a flask followed by THF (80 Vol, 150 mL) and piperidine (2.3 g, 28 mmol, 20 eq). The resulting si urn was stirred under a nitrogen atmosphere at ambient temperature. After stirring for 3 hours, the solvent was reduced via rotary' evaporation and acetonitrile (100 mL) was added to the thick paste in the flask and stirred. After stirring for 2 hours, the slurry was filtered, and the wet cake was washed with acetonitrile (100 mL). The wet solid was transferred to a vacuum oven and dried under vacuum at an ambient temperature. After dry ing for 3 days, 1.3 g of 30 was isolated as an off-white solid (81% yield). FIG. 29 shows the 'l l NMR (400 MHz, CDC13) of compound 30. FIG. 30 shows the LCMS (ES+) chromatograph of compound 30. LCMS m / z found: 1140.8298 (M+H+).(P101782W001)Fmoc-Phe-Gly-Gly-Phe-Gly-Gly-Tag5P-S (31):

[0231] Fmoc-PheGlyGly-OH (0.51 g, 1.0 mmol, 1.3 eq) was added to a flask followed by THF (30 Vol, 27 mL), DIC (0.22 g, 1.7 mmol, 2.2 eq), and Oxyma (0.20 g, 1.4 mmol, 1.8 eq). The resulting slurry was stirred for 10 minutes before H-Phe-Gly-Gly-Tag5P-S 30 (0.90 g, 0.79 mmol, 1.0 eq) was added followed by DMF (3 Vol, 2.7 mL). After stirring for 16 hours, the reaction mixture was concentrated via rotary' evaporation to afford a yellow' gel. Acetone (20 mL) and acetonitrile (15 mL) w ere added to the gel and the resulting slurry w as stirred for 4 hours before it was filtered. The filter cake was rinsed with acetone (10 mL) then acetonitrile (20 mL) and the gel-like solid was transferred to a vacuum oven and dried overnight under vacuum at ambient temperature. After drying for ~ 18 hours, 1.09 g of 31 was isolated as an off-white solid (85% yield). FIG. 31 shows the 'H NMR (400 MHz, CDC13) of compound 31.H-Phe-Gly-Gly-Phe-Gly-Gly-Tag5P-S (32):

[0232] Fmoc-Phe-Gly-Gly-Phe-Gly-Gly-Tag5P-S 31 (0.95 g, 0.58 mmol. 1.0 eq) was added to a flask followed by THF (30 Vol, 30 mL), DMF (3 Vol, 3 mL), and piperidine (0.98 g, 11.6 mmol, 20 eq). The resulting sluny was then stirred under at ambient temperature overnight. After stirring for 18 hours, the solvent was reduced via rotary evaporation to afford a thick oil. Acetonitrile (60 mL) was added to the oil in the flask and stirred. After stirring for 4 hours, the resulting sluny' was filtered and the wet cake was washed with acetonitrile (10 mL). The wet solid was transferred to a vacuum oven and dried under vacuum at an ambient temperature. After dry ing for 3 days, 0.78 g of 32 was isolated as an off-white solid (95% yield). FIG. 32 shows the1H NMR (400 MHz, CDC13)(P101782W001) of compound 32. FIG. 33 shows the LCMS (ES+) chromatograph of compound 32.LCMS m / z found: 1401.9411 (M+H+).H-Phe-Gly-Gly-Phe-Gly-Gly-OH (33):

[0233] H-Phe-Gly-Gly-Phe-Gly-Gly-Tag5P-S 32 (0.040 g, 0.029 mmol, 1.0 eq) was added to a vial followed by a solution of TFA / H2O (95:5. 12.5 Vol, 0.5 mL). A pink solid precipitated once the solvent was added and the resulting slurry was stirred for 1.5 hours at ambient temperature. After stirring for 1.5 hours, the liquid was decanted and MTBE (5 mL) was added to it, resulting in the precipitation of a pale blue solid. After stirring for 3 hours, the slurry7was filtered and the isolated gel-like solid was washed with MTBE (2 mL) followed by DCM (2 mL). The wet solid was transferred to a vacuum oven and dried overnight under vacuum at ambient temperature. After drying for ~18 hours, 0.010 g of 33 was isolated as pale blue solid (67% yield). FIG. 34 shows the 'H NMR (400 MHz, D2O) of compound 33. 'H NMR (400 MHz, D2O) 5 7.33 - 7.02 (m, 10H), 4.50 - 4.43 (m, 1H), 4.12 (t, J = 7.3 Hz, 1H), 3.79 (d, J = 3.9 Hz, 2H), 3.74 (d, J = 1.4 Hz, 2H), 3.72 - 3.68 (m, 2H), 3.67 - 3.65 (m, 1H), 3.07 - 2.95 (m, 3H), 2.87 (d, J = 8.7 Hz, 1H). FIG. 35 shows the LCMS (ES+) chromatograph of compound 33. LCMS m / z found: 541.2 (M+H+).

[0234] 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

(P101782W001)CLAIMS1. A selectively soluble support compound represented by Formula (I):(I), wherein,Ri is a straight or branched C6-C40 aliphatic group;R2 is H or a straight or branched C1-C40 aliphatic group; andY is selected from the group consisting of H, succinate, -C(O)-X. -C1-C10 alkyl- C(O)-X, -C(0)-(Ci-Cio alkyl)-C(O)-X, and -C(O)-aryl-C(O)-X, wherein X is Cl, Br, I, F, imidazole, or other 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 H.

4. The selectively soluble support compound of claim 1, wherein Ri is C18H37.

5. The selectively soluble support compound of claim 4, wherein R2 is methyl.

6. The selectively soluble support compound of claim 1, wherein R2 is a straight or branched C1-C40 aliphatic group.

7. The selectively soluble support compound of claim 1, wherein Y is succinate.

8. The selectively soluble support compound of claim 1, wherein Y is selected from the group consisting of -C(O)-X, -C1-C10 alkyl-C(O)-X, -C(0)-(Ci-Cio alkyl)-C(O)- X, and -C(O)-aryl-C(O)-X; and X is imidazole.

9. The selectively soluble support compound of claim 1, wherein the compound is10. The selectively soluble support compound of claim 1. wherein the compound is(P101782W001)11. 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):wherein,Ri is a straight or branched C6-C40 aliphatic group;R2 is H or a straight or branched C1-C40 aliphatic group; andY is selected from the group consisting of H, succinate, -C(O)-X, -, -C1-C10 alkyl-C(O)-X, -C(0)-(Ci-Cio alkyl)-C(O)-X, and -C(O)-aryl-C(O)-X, wherein X is Cl, Br, I, F. imidazole, or other leaving group.

12. The method of claim 11. wherein Y is selected from the group consisting of -C(O)-X, -, -C1-C10 alkyl-C(O)-X, -C(0)-(Ci-Cio alkvl)-C(O)-X, and -C(O)-aryl-C(O)-X; and X is imidazole.(P101782W001)13. The method of claim 11, wherein the selectively soluble support compound14. The method of claim 11, wherein the selectively soluble support compound represented by Formula (I) is15. The method of claim 11, which 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.

16. The method of claim 15, 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(P101782W001) 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.

17. The method of claim 16, further comprising a fourth step including converting the phosphite 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.

18. The method of claim 16, further comprising a fourth step including converting the phosphite diester bond of the oligomer into a phosphodiester bond, a thiophosphodiester bond, a boranophosphodiester bond or an aminophosphodiester bond.

19. The method of claim 18, 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.

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

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

22. A selectively soluble support compound represented by Formula (II):(P101782W001)wherein,Ri is a straight or branched C6-C40 aliphatic group;R2 is H or a straight or branched C1-C40 aliphatic group;R3 is H or -O-alkyl:R4 is H or -O-alkyl; andY is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F, imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl.

23. The selectively soluble support compound of claim 22, wherein Y is OH.

24. The selectively soluble support compound of claim 22, wherein R2 is H.

25. The selectively soluble support compound of claim 22, wherein Ri is C18H37.

26. The selectively soluble support compound of claim 22, wherein R3 is -O-methyl and R4 is H.

27. The selectively soluble support compound of claim 22, wherein R3 is H and R4 is - O-methyl.

28. The selectively soluble support compound of claim 22, wherein the compound is(P101782W001)29. The selectively soluble support compound of claim 22, wherein the compound is30. A liquid phase method of producing a peptide comprising the following steps;(1) obtaining a C-protected amino acid or C-protected peptide, comprising condensing a compound of Formula (II):wherein,Ri is a straight or branched C6-C40 aliphatic group;R2 is H or a straight or branched C1-C40 aliphatic group;R3 is H or -O-alkyl;R4 is H or -O-alkyl; andY is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F. imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl. with a C-terminal of N-protected amino acid or N-protected peptide;(2) removing a temporary protecting group of a N-terminal of the amino acid or peptide obtained in the above-mentioned step;(3) condensing the N-terminal of the amino acid or peptide obtained in the above- mentioned step with N-protected amino acid or N-protected peptide; and(4) precipitating the peptide obtained in the above-mentioned step.(P101782W001)31. The method of claim 30, wherein Y is selected from the group consisting of -C(O)-X, -Ci-Cio alkyl-C(O)-X, -C(0)-(Ci-Cio alkyl)-C(O)-X. and -C(O)-aiyl-C(O)-X; and X is imidazole.

32. The method of claim 30, wherein the selectively soluble support compound represented by Formula33. The method of claim 30, wherein the selectively soluble support compound represented by Formula34. The liquid phase method of claim 30 wherein Y is -OH.

35. The liquid phase method of claim 30 wherein Ri is C18H37.

36. The liquid phase method of claim 30 wherein one of R3 and R4 is -O-Methyl.

37. The liquid phase method of claim 30, further comprising a step of cleaving the formed peptide from the compound of Formula (II).

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