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

Abstract

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

Description

SELECTIVELY SOLUBLE CITRIC ACID-DERIVED SUPPORTS FOR LIQUIDPHASE PEPTIDE AND OLIGONUCLEOTIDE SYNTHESISTECHNICAL FIELD

[0001] The field of the disclosure relates generally to amino acids, peptides, proteins, and nucleic acids for pharmaceutical industry7and, more specifically, using selectively soluble supports for liquid phase organic synthesis of peptides and oligonucleotides.BACKGROUND OF THE INVENTION

[0002] The present invention relates to selectively soluble supports for liquid-phase peptide and oligonucleotide synthesis. The selectively soluble supports (also referred to herein as "lags") 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 peptide or oligonucleotide synthesis, particularly liquid phase synthesis, and methods of synthesis using such a compound.

[0003] Despite the widespread adoption of solid-phase synthesis of peptides and oligonucleotides 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.

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

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

[0006] In an attempt to perform reactions in a homogeneous liquid phase while 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.

[0007] 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 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.

[0008] 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.

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

[0010] Still, there is a need in the art for new selectively soluble supports for liquid phase organic synthesis of peptides and oligonucleotides which are superior in broad utility and stability, which are useful as a protecting group (anchor) for peptide and oligonucleotide synthesis in the liquid phase. Prior art supports are typically limited to the synthesis of peptides consisting of only 6-8 amino acids, whereas the citric acid-derived supports disclosed in the present application enable the synthesis of longer peptide chains, including up to 10 amino acids, thereby addressing the limitations of existing materials.BRIEF SUMMARY OF THE INVENTION

[0011] In one aspect, disclosed herein is a selectively soluble support compound derived from citric acid, the selectively soluble support compound represented by Formula (I):wherein R1is a straight or branched C6-C40alky l or aliphatic group; R2is H or O-alkyl; R3is 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-C30alkyl group, or a C7-C30aralkyl group, wherein the alkyl portion of the C7-C30aralkyl group is a straight or branched C1-C30alky l.

[0012] In another aspect, disclosed herein is a liquid phase method of producing a peptide comprising the following steps; obtaining C-protected amino acid or C-protected peptide, comprising condensing a compound of Formula (I):wherein R1is a C6-C40straight or branched alkyl or aliphatic group; R2is H or O-alkyl; R3is 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 another leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30alkyl group, or a C7-C30aralkyl group, wherein the alkyl portion of the C7-C30aralkyl group is a straight or branched C1-C30alkyl, with the C- terminal of N-protected amino acid or N-protected peptide; removing a temporary protecting group of the N-terminal of the amino acid or peptide obtained in the above- mentioned step; 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 precipitating the peptide obtained in the above-mentioned step.

[0013] In yet another aspect, disclosed herein is a liquid phase method of producing an oligonucleotide comprising a step of subjecting a 5'-hydroxyl group or 3'-hydroxyl group of a nucleoside or oligonucleotide having a selectively soluble support in at least one location selected from the group consisting of 2'-position, 3'-position, 5'-position and a nucleobase moiety and having a 5'-hydroxyl group or a 3'-hydroxyl group, to H- phosphonation to convert the 5'-hydroxyl group or the 3'-hydroxyl group into an H- phosphonated form, wherein the selectively soluble support is a compound represented by Formula (I):wherein R1is a straight or branched C6-C40alkyl or aliphatic group; R2is H or O-alkyl; R3is H or O-alkyl; and ¥ 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-C30alkyl group, or a C7-C30aralkyl group, wherein the alkyl portion of the C7-C30aralkyl group is a straight or branched C1-C30alkyl.

[0014] Compounds of Formula (I) can be used as a both a support for peptide and 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 the carbonyl group is protected by a compound of Formula (I) is characterized by tending to be in a liquid slate and its high solubility in a 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.

[0015] 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 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.

[0016] The method of synthesizing a peptide using a compound of Formula (I) 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.

[0017] (1) The compound of Formula (I) is condensed with the C-terminal carboxy group of an N-protected amino acid or N-protected peptide in a soluble solvent to obtain an N- protected C-protected amino acid or N-protected C -protected peptide in which the C- terminal is protected by the compound of Formula (I).

[0018] (2) The protecting group on the N-terminal of the resulting N-protected C- protected amino acid or N-protected C-protected peptide is removed to obtain a C- protected amino acid or C-protected peptide.

[0019] (3) An N-protected amino acid or N-protected peptide is condensed with the N- terminal of the resulting C-protected amino acid or C-protected peptide to obtain an N- protected C-protected peptide.

[0020] (4) The protecting groups on the N-terminal and the protecting group on the C- terminal of the resulting N-protected C-protected peptide are removed to obtain the target peptide.

[0021] This procedure using the selectively soluble support compounds of Formula (I) can also be used to synthesize an oligonucleotide in a liquid phase synthesis.

[0022] The tags of Formula (I) allow for easy in-process NMR analysis for confirmation.

[0023] The embodiments and features of the present invention can be used alone or in combinations with each other.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings.

[0025] FIG. 1 is an1H NMR spectrum of compound 3 of the Examples in CDCl3;

[0026] FIG. 2 is an1H NMR spectrum of compound 5 of the Examples in CDCl3;

[0027] FIG. 3 is an1H NMR spectrum of compound 7 of the Examples in CDCl3;

[0028] FIG 4 is an1H NMR spectrum of TAG 1 in CDCl3;

[0029] FIG. 5 is an1H NMR spectrum of TAG 1 in CDCl3after heptane purification;

[0030] FIG. 6 is an1H NMR spectrum of tripeptide 13A of the Examples in D2O;

[0031] FIG. 7 is a13C NMR spectrum of tripeptide 13A of the Examples in D2O;

[0032] FIG. 8 is an LCMS (ES+) chromatograph of tripeptide 13A of the Examples;

[0033] FIG. 9 is an LCMS (TIC, ESI) chromatograph of tripeptide 13A of the Examples(purity);

[0034] FIG. 10 is the1H NMR spectrum of the hexapeptide 19A of the Examples in D2O;

[0035] FIG. 11 is the13C NMR spectrum of hexapeptide 19A of the Examples in D2O;

[0036] FIG. 12 is the LCMS (ES+) chromatograph of hexapeptide 19A of the Examples;

[0037] FIG. 13 is the LCMS (TIC, ESI) chromatograph of hexapeptide 19 A (purity) of the Examples;

[0038] FIG. 14 is the1H NMR spectrum of the hexapeptide 27 A of the Examples in D2O;

[0039] FIG. 15 is the13C NMR spectrum of hexapeptide 27A of the Examples in D2O;

[0040] FIG. 16 is the LCMS (ES+) chromatograph of hexapeptide 27A of the Examples; and

[0041] FIG. 17 is the LCMS (TIC. ESI) chromatograph of hexapeptide 27A (purity) of the Examples.DETAILED DESCRIPTION OF THE INVENTION

[0042] 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 descnbed invention.Definitions

[0043] 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.

[0044] 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.

[0045] As used herein, the term "‘selectively soluble support" or "tag" is a compound that, when reacted with a peptide or a nucleotide, protects a reactive site on the peptide or nucleotide and supports the elongation of the peptide or nucleotide and can be solubilized or precipitated by changing the composition of the solvent in which the elongation reactions occur.

[0046] 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 carbon atoms, or 2 or 3 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, t- butyl, isobutyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), hexyl, isohexyl, heptyl, octyl, nonyl, 4,4-dimethylpentyl, 2,2,4-trimethylpentyl, decyl, undecyl, dodecyl, 2-methyl-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- 1 -propyl. 3-methyl-l-pentyl. 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.

[0047] As used herein, the term “alkylamino” means an amino group substituted by at least one alky l group. In some embodiments, the alkyl group is a lower alkyl group having from 1 to 6 carbon atoms. Alkylamino groups include, but are not limited to, -NHCH2CH3, -NH(CH2)2CH3, -NH(CH2)3CH3, -NH(CH2)4CH3, and -NH(CH2)5CH3, and the like.

[0048] As used herein, the term “amino’' means -NH2.

[0049] As used herein, the term “aryl” means a monocyclic, bicyclic, or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbon. In some embodiments, the aryl group has from 6 to 20 carbon atoms or from 6 to 10 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and tetrahydronaphthyl, and the like.

[0050] As used herein, the term “aralkyl” or “arylalkyl” refers to a monovalent alkyl group substituted with one or more aryl groups. In certain embodiments, the aralkyl has from 7 to 30, from 7 to 20, or from 7 to 16 carbon atoms. Examples of aralky l groups include, but are not limited to, benzyl, 1 -phenylethyl, 2-phenylethyl, and 3-phenylpropyl. In certain embodiments, the aralkyl is optionally substituted with one or more.

[0051] The term “halogen”, “halide” or “halo” refers to fluorine, chlorine, bromine, and / or iodine.

[0052] 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 l'-position of a sugar (e.g., ribose or deoxyribose wherein the carbon atoms at 2 -position and carbon atom at 4-position of 2-deoxyribose, ribose, ribose ring or deoxyribose ring are bonded by a divalent organic group, and the like) by N-glycosidation. Examples of the ribose or deoxyribose wherein the carbon atom at 2 -position and carbon atom at 4-position of the ribose ring or deoxyribose ring are bonded by a divalent organic group include the following compounds:

[0053] As used herein, the term "‘nucleotide-’ means a compound wherein a phosphoric acid group is bonded to a nucleoside.

[0054] 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).

[0055] 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.

[0056] 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 foridentifying discrete activities or ingredients and the recited lettering can be arranged in any sequence.

[0057] 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

[0058] The compounds of the present invention are selectively soluble support compounds derived from citric acid and are represented by Formula (I):wherein R1is a straight or branched C6-C40alky l or aliphatic group; R2is H or O-alkyl; Rs 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-C30alkyl group, or a C7-C30aralkyl group, wherein the alkyl portion of the C7-C30aralkyl group is a straight or branched C1-C30alky l.

[0059] A compound represented by Formula (I) of the present invention is bonded to a compound to be protected via group Y.

[0060] 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.

[0061] Examples of the “alkyl group” for R include a 61-30 alkyl group, preferably a C1-10alkyl group, more preferably a C1-6alkyl group. Preferable examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl tert- butyl, and the like, and particularly preferred are methyl and ethyl.

[0062] In the present specification, examples of the “aralkyl group” for R include a 67-30 aralkyl group. Preferred is a C7-20aralkyl group, more preferred is a C7-16aralkyl group (a C6-40aryl-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.

[0063] As R, a hydrogen atom, a C1-6alkyl group or a C7-16aralkyl group is preferable, a hydrogen atom, methyl, ethyl or benzyl is more preferable, and a hydrogen atom is particularly preferable.

[0064] In some embodiments, Y is -OH. In other embodiments, Y is -OH and R1is C18H37.

[0065] In other embodiments, Y is -NHR. In embodiments when Y is -NHR, R is H, a C1-C30alkyl group, or a C7-C30aralkyl group. In embodiments wherein R is C1-C30alkyl, R is preferably methyl. In embodiments wherein R is C7-C30aralkyl, R is preferably methyl.

[0066] In some embodiments, R is H.

[0067] Examples of the “aralky l group” for R include a C7-30aralkyl group. Preferred is a C7-20 aralkyl group, more preferred is a C7-16aralkyl group (a C6-40aryl-C1-6alkyl group). Preferable examples include benzy l, 1 -phenylethyl, 2 -phenylethyl, 1 -phenylpropyl, naphthylmethyl, 1 -naphthylethyl, 1 -naphthylpropyl, and the like, and particularly preferred is benzy l.

[0068] The “C6-C40aliphatic group” for R1means a monovalent organic group having an aliphatic hydrocarbon group in the molecular structure. The Rl groups are sometimes referred to herein as ‘"tails,” whose lengths can be selected to impact solubility of the compound.

[0069] The “aliphatic group” is an aliphatic hy drocarbon 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.

[0070] 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 isobuty l group, a sec-buty l group, a tert-buty l group, a pentyd group, a hexyl group, an octy l group, a decyl group, a dodecyl group, a lauryl group, a tridecyl group, a myristyl group, a cetyl group, a stearyl group, an aralkyl group, a behenyl group, an oleyl group, an isostearyl group and the like and divalent groups derived therefrom. In preferred embodiments, R1is a stearyl group, C18H37.

[0071] In some embodiments of Formula (I), R2and R3are not the same. In some embodiments, R2is H and R3is O-alkyl. In some embodiments, R2is O-alkyl and R3is H. In other embodiments, R2is O-methyl and R3is O-ethyl.

[0072] In some embodiments of Formula (I), wherein R2is -OMe and R3is H, an example of a selectively soluble support compound of Formula (I) is Formula (II):

[0073] In some embodiments of Formula (I), wherein R2is H and R3is -OMe, an example of a selectively soluble support compound of Formula (1) is Formula (III):Production Method of Compounds of Formula (I)

[0074] 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 in Example 1 for the compound,trioctadecy l 2-((4-(4-(hydroxymethyl)-2-methoxyphenoxy)butanoyl)oxy)propane-l,2,3- tricarboxylate. Notably, the starting compound is citric acid, which is readily available and is inexpensive.Use as a Selectively Soluble Support in Organic Synthesis for Peptides

[0075] 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.

[0076] As the peptide production method including the peptide synthesis method of the present invention, for example, steps as shown below are exemplified.

[0077] 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 hydroxy group, a diolgroup, and an amino group, and examples thereof include amino acids, peptides, saccharides, proteins, nucleotides, various medicinal compounds and agrochemical compounds, and various polymers and dendnmers.

[0078] 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 (Tag) 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.

[0079] The method of synthesizing a peptide using a compound of Formula (I) 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.

[0080] step (1) a step of dissolving the compound of the present invention in soluble solvent (dissolution step),

[0081] 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),

[0082] step (3) a step of precipitating the bonded product obtained in the above- mentioned step (precipitation step), and

[0083] 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).

[0084] Step (1) (Dissolution Step)

[0085] In this step, the selectively soluble support compound of Formula (I) is dissolved in a soluble solvent.

[0086] 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 (I) 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.

[0087] Step (2) (Bonding Step)

[0088] In this step, the compound of Formula (I) dissolved in a soluble solvent, which is obtained in the above-mentioned step (1) is bonded to a reaction substrate.

[0089] 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.

[0090] When V 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 dimethy laminopy ri dine cataly s t.

[0091] When V is a NHR group, for example, 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), l-hydroxy-7-azabenzotriazole (HO At) and the like.

[0092] 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.

[0093] Examples of the condensing agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIG), 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'-tetramethyl uronium tetrafluoroborate (TBTU), l-[bis(dimethylamino)methylene]-5-chloro- IH-benzotriazolium 3-oxide hexafluorophosphate (HCTU). O-benzotriazole-N,N,N'.N'- tetramethyl uronium hexafluoroborate (HBTU), and the like.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.1H NMR 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.

[0098] Step (3) (Precipitation Step)

[0099] 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 areused for precipitation. Isolation by filtration and further washing of the isolated solid with additional portions of the polar solvents ) should then afford the clean product.

[0100] Step (4) (Deprotection Step)

[0101] In this step, the selectively soluble support compound of Formula (I) is finally cleaved / deprotected from the bonded product or resulting product isolated by precipitation in the above-mentioned step (3) to give the object compound.

[0102] When V 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 ¥ 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.

[0103] 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, dichloromethane and THF, having an acid concentration of from about 0. 1% to about 97%.

[0104] 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.

[0105] 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.

[0106] By utilizing the above-mentioned steps, a peptide can be produced. The compound of Formula (I) 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 (I) wherein ¥ 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.

[0107] A method of producing a peptide utilizing the above-mentioned steps, comprising the following steps;

[0108] (1) a step of obtaining C-protected amino acid or C-protected peptide, comprising condensing a compound of Formula (I) with the C -terminal of N-protected amino acid or N-protected peptide (C -terminal protection step),

[0109] (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),

[0110] (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

[0111] (4) a step of precipitating the peptide obtained in the above-mentioned step (precipitation step).

[0112] Step (1) (C-Terminal Protection Step)

[0113] In this step, the compound of Formula (I) is condensed with the C -terminal of N- 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.

[0114] 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)).

[0115] The condensation reaction of the compound of Formula (I) 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.

[0116] 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 benzylic compound of the present invention.

[0117] The reaction temperature is generally -10 °C to 40 °C, preferably 0 °C to 30 °C.The reaction time is generally 1-70 hr.

[0118] Step (2) (N-Terminal Deprotection Step)

[0119] 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.

[0120] As the temporary protecting group of the N-terminal, the below-mentioned aminoprotecting groups generally used in the technical field of peptide chemistry7and 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.

[0121] 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.

[0122] 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.

[0123] Step (3) (Peptide Chain Elongation Step)

[0124] 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.

[0125] 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.

[0126] Step (4) (Precipitation Step)

[0127] This step is performed in the same manner as in the precipitation step in the above-mentioned precipitation step.

[0128] 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:

[0129] (5) a step of removing the temporary protecting group of N-terminal of the peptide obtained in the precipitation step,

[0130] (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

[0131] (7) a step of precipitating the peptide obtained in the above-mentioned step.

[0132] Step (5)

[0133] The step is performed in the same manner as in the deprotection step of N- terminal in step (2).

[0134] Step (6)

[0135] This step is performed in the same manner as in the peptide chain elongation step of step (3).

[0136] Step (7)

[0137] This step is performed in the same manner as in the precipitation steps described above.

[0138] 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 (I), 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 / cleavage of the anchor of the present invention.

[0139] 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, crystallization, chromatography and the like, to isolate and purify a peptide as the final target.

[0140] 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.

[0141] 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.

[0142] In a final step (excluding any purification steps), the formed peptide is cleaved / deprotected 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.

[0143] 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.

[0144] (Composition Changing Means)

[0145] 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 C-carrier protected peptide are used without particular restriction. The preferable means for changing a composition of a solution includes, for example, means in which a poor solvent is added to a solution dissolving a C-carrier protected peptide as it is or a poor solvent is added after concentrating the solvent of the solution, tocause crystallization. Here, concentrating means to distill off a part or all of the solvent. Thereafter, the precipitated crystal can be separated, for example, by filtration and centrifugal separation. Impurities separated together with the crystal can be removed completely from the crystallized C-carrier protected peptide, preferably by washing the separated cry stal with an organic solvent.

[0146] The poor solvent used in the present invention means a solvent in which a C- protected amino acid amide is poorly soluble, that it, a C-protected amino acid amide is not dissolved easily or is not dissolved. For C-protected amino acid amide is not dissolved easily or is not dissolved, the poor solvent may advantageously be a solvent which is liquid at normal temperature with which the solubility of a C-protected amino acid amide is less than 1% by mass at 25 °C, and preferable are acetonitrile, any proportioned aqueous acetonitrile, methanol, any proportioned aqueous methanol and water.

[0147] This procedure using the selectively soluble support compounds of Formula (I) can also be used to synthesize an oligonucleotide in a liquid phase synthesis.Use as a Selectively Soluble Support in Organic Synthesis of Oligonucleotides

[0148] 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-mer oligonucleotide”) to produce an oligonucleotide (hereinafter, also written as the "(n+p)-mer oligonucleotide”) protected with a selectively soluble support of Formula (I). For oligonucleotide synthesis, the Y portion of the compounds of Formula (I) is preferably selected from the group consisting of -OH, - COOH, O-C(O)-X, wherein X is Cl, Br, I, F, imidazole, and 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. This is to accommodate an ester linkage to the 3’ OH group on a nucleoside via an O- C(O)- linkage. The n-mer oligonucleotide means an oligonucleotide in which n nucleoside units are linked together via a phosphorus-containing group. When n is 1, the n-mer oligonucleotide is understood as a nucleoside. The same applies to the p-mer oligonucleotide.

[0149] 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.

[0150] Further, the oligonucleotide production method includes at least one elongation reaction cycle including the above H-phosphonation step.

[0151] 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).

[0152] Step (a)

[0153] 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 al the elongation terminal a hydroxyl group protected with a temporary protecting group, to remove the temporary protecting group to form a hydroxyl group.

[0154] Step (b)

[0155] 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.

[0156] Step (c)

[0157] 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.

[0158] Step (d)

[0159] 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.

[0160] Here, the amino group in the aminophosphodiester bond is unsubstituted or is substituted with one or two C1-6alkyl groups.

[0161] The phosphodiester bond protected with a basic protecting group is a bond formed by substituting a phosphodi ester bond with, for example, a C1-40alkyl group, a C3-6cycloalkyl group, a C6-10aryl group, a 5-10 membered heteroaryl group, an aralkyl group or a heteroaralkyl group in place of the hydrogen atom of the single hydroxyl group. Here, the C1-6alkyl group is unsubstituted, or is substituted with a substituent such as a halogen atom or a cyano group. The C3-6cycloalkyl group, the C6-10aryl group, the 5-10 membered heteroaryl group, the aralkyl group and the heteroaralky l group are unsubstituted, or are substituted with a substituent such as a C1-6alkyl group, a halogen atom or a cyano group.

[0162] The thiophosphodiester bond protected with a basic protecting group is a bond formed by substituting a thiophosphodi ester bond with, for example, a C1-40alkyl group, a C3-6cycloalkyl group, a C6-10aryl group, a 5-10 membered heteroaryl group, an aralkyl group or a heteroaralky l group in place of the hydrogen atom of the single thiol group. Here, the C1-40alkyl group is unsubstituted, or is substituted with a substituent such as a halogen atom or a cyano group. The C3-6cycloalkyl group, the C6-10ary l 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 C1-6alkyl group, a halogen atom or a cyano group.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] Step (e)

[0168] 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.

[0169] 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.

[0170] 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).

[0171] 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).

[0172] 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.

[0173] 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.

[0174] 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.

[0175] The oligonucleotide production method may further include Step (1), in which case the desired oligonucleotide can be isolated and produced.

[0176] Step (f)

[0177] Step (f) is a deprotection step of removing all of the basic protecting group, the temporary' protecting group and the selectively soluble support of Formula (I) in the oligonucleotide obtained by Step (a) through Step (e).

[0178] 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.

[0179] 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.

[0180] 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 16 7 trioctadecyl 2-((4-(4-(hydroxymethyl) -2-methoxyphenoxy)butanoyl)oxy) propane- 1 ,2,3-tricarboxylateStearvl Citrate Triester (3)1 2 3

[0181] Citric acid 1 (5.0 g, 26.08 mmoL, 1.0 eq), stearyl alcohol 2 (25.5 g, 94.27 mmoL, 3.6 eq), and / i-TsOH • H2O (498.6 mg. 2.62 mmoL, 0. 1 eq) were placed to a r.-b. flask. Toluene (50 mL. 10 vol.) was added. The mixture was stirred at 140 °C (hot plate) under N2 for 19 h. It was let to cool down over 1 h. EtOH (150 mL, 30 vol.) was added. The mixture w as stirred at RT for 15 min. It was filtered and w ashed with EtOH (3 x 60 mL, 3 x 12 vol.). The solid was dissolved in EtOH (250 mL, 50 vol.) at 62 °C (hot plate). It was allowed to cool down to RT over 3 h. It was filtered and washed with EtOH (3 x 60 mL, 3 x 12 vol.). It was dried in a vacuum oven at RT overnight. It was isolated 22.8 g (92 %) of white solid. FIG. 1 show s the1H NMR of compound 3 in CDCI3.Stearyl Citrate Bromobutanoate (5)

[0182] Stearyl citrate triester 3 (19.8 g, 20.85 mmoL, 1.0 eq) was placed to a r.-b. flask. Bromobutyryl chloride 4 (24.4 mL, 210.78 mmoL, 10.1 eq) was added. The mixture was stirred neat at 75 °C (hot plate) under N2for 23 h. It was let to cool down over 5 min. EtOH (200 mL, 10 vol.) was added. It was stirred at 75 °C (hot plate) under N2 for 5 min. It was let to cool down to RT over 1 h. Stearyl citrate bromobutonoate 5 was filtered and washed with EtOH (3 x 60 mL, 3 x 3 vol.). It was dried in a vacuum oven at RT overnight. It was isolated 22.2 g (97 %) of white solid. FIG. 2 shows the1H NMR spectrum of compound 5 in CDCl3.Stearvl Citrate Vanillin Butanoate (7)Stearyl citrate bromobutanoate 5 (10.1 g, 9.16 mmoL, 1.0 eq), vanillin 6 (4.2 g,27.43 mmoL, 3.0 eq), and CS2CO3 (5.4 g, 16.45 mmoL, 1.8 eq) were placed to a r.-b. flask.DMF (200 mL. 20 vol.) was added. The mixture was stirred at 45 °C (hot plate) under N2 for 22 h. It was let to cool down to RT over 1 h. EtOH (300 mL, 30 vol.) was added. It was stirred at 0 °C for 30 min. It was filtered and washed with H2O (3 x 50 mL, 3 x 5 vol.) andEtOH (2 x 50 mL, 2 x 5 vol.). It was dried in a vacuum oven at RT overnight. Steary l citrate vanillin butanoate 7 was isolated 9.6 g (90 %) of light-yellow solid containing ~ 20 % of CIS impurity. FIG. 3 shows therH NMR spectrum of compound 7 in CDCl3.Stearvl Citrate Butanoate Vanillvl Alcohol (TAG 1)

[0183] Stearyl citrate vanillin butanoate 3 (4.5 g, 3.87 mmoL. 1.0 eq) was placed to a r.-b. flask. CH2CI2(50 mL, 11 vol.) and MeOH (40 mL, 9 vol.) were added. The mixture was placed to an ice bath and cooled down over 10 min. NaBH4(372.9 mg, 9.86 mmoL, 2.5 eq) was added to citrate al 0 °C. The mixture was stirred at 0 °C under N2for 1 h. Secondportion of NaBH4(370.7 mg, 9.80 mmoL, 2.5 eq) was added at 0 °C. The mixture was removed from the ice bath and stirred at RT for another 1 h. H2O (4 mL, 1 vol.) was added. It w as stirred at RT for 5 min. CH2CI2was evaporated at 20 °C (350 mbar). EtOH (60 mL.13 vol.) was added. The slurry was stirred at 0 °C for 1 h. It was filtered and washed with H2O (3 x 15 mL, 3 x 3 vol.) and cold EtOH (2 x 15 mL, 2 x 3 vol.). It was dried in a vacuum oven at RT overnight. It was isolated 3.5 g (78 %) of light-yellow solid containing ~ 20 % of C18 impurity. FIG. 4 is the1H NMR spectrum of TAG 1 in CDCl3.TAG 1 (Purification)

[0184] To remove the Cl 8 impurity, stearyl citrate butanoate vanillyl alcohol (TAG 1) was further purified with heptane following one of the two following procedures.

[0185] Hot Recry stallization

[0186] Stearyl citrate butanoate vanillyl alcohol (1.1 g, 0.94 mmoL, 1.0 eq) was mixed with heptane (22 mL, 20 vol.). The mixture was heated to 70 °C (hot plate) under N2 until all solids dissolved. It was let to cool down to RT over 2 h. It was filtered. It was dried in a vacuum oven at RT overnight. It was isolated 0.6 g (54 %) of light-yellow solid containing ~ 5 % of Cl 8 impurity.

[0187] Sonication

[0188] Stearyl citrate butanoate vanillyl alcohol (4.85 g. 4.14 mmoL, 1.0 eq) was mixed with heptane (90 mL, 20 vol.). The mixture was sonicated at RT for 5 min. It was filtered. It w as dried in a vacuum oven at RT overnight. It was isolated 2.9 g (60 %) of light-yellow solid containing ~ 5 % of C18 impurity. FIG. 5 shows the 'H NMR of TAG 1 in CDCl3after heptane purification.Example 2; Peptide Syntheses Using Tag 1A. Synthesis of 3-mer Peptide

[0189] TAG 1 (8.10 g, 6.91 mmoL, 1.0 eq), Fmoc-Gly-OH (3.08 g, 10.36 mmoL, 1.5 eq), and DMAP (166.9 mg. 1.37 mmoL, 20 mol %) were placed to a r.-b. flask. CH2CI2(160 mL, 20 vol.) was added followed by DIC (1.6 mL, 10.33 mmoL, 1.5 eq). The mixture was stirred at RT for 2 h. The solvent was evaporated. EtOH (160 mL, 20 vol.) was added, and the mixture was stirred at RT for 15 min. It was filtered and washed with EtOH (3 x 40 mL, 3 x 5 vol.). It was dried in a vacuum oven at 30 °C overnight. It was isolated 9.05 g (90 %) of light-yellow solid (8).

[0190] Fmoc-Gly-TAG 8 (708.3 mg. 0.49 mmoL, 1.0 eq) was placed to a r.-b. flask. CH2CI2(5 mL, 7 vol.) was added. Piperidine (480 pL, 4.86 mmoL, 10.0 eq) was added.The mixture was stirred at RT for 1 h. Solvent was evaporated. MeCN (11 mL, 15 vol.) was added. It was stirred at RT for 15 min. It was filtered and washed with MeCN (3 x 5 mL, 3 x 7 vol.). It was dried in a vacuum oven at 30 °C overnight. It was isolated 536.7 mg (89 %) of beige solid (9).Fmoc-Val-Gly-TAG (10)

[0191] FLN-Gly-TAG 9 (400.0 mg, 0.32 mmoL, 1.0 eq) was placed to a r.-b. flask. CH2CI2(1 mL, 2.5 vol.) was added. Fmoc-Val-OH (226.9 mg, 0.67 mmoL. 2.1 eq) and Oxyma Pure (93. 1 mg. 0.65 mmoL, 2.0 eq) were placed to a vial. CH2CI2(1 mL. 2.5 vol.) and THF (1 mL, 2.5 vol.) were added to the vial followed by DIC (100 pL, 0.65 mmoL, 2.0 eq). The solution turned yellow. It was let to activate for 2 min and then transferred to the flask with compound 9. The mixture was stirred at RT for 1 h. The solvent was evaporated. MeCN (8 mL, 20 vol.) was added, and the slurry was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 4 mL, 3 x 10 vol.). It was dried in a vacuum oven at 30 °C overnight. It was isolated 473.9 mg (95 %) of beige solid (10).HzN-Val-Glv-TAG (11)

[0192] Fmoc-Val-Gly-TAG 10 (325.5 mg. 0.21 mmoL, 1.0 eq) was placed toa r.-b. flask. CH2CI2(2 mL. 6 vol.) was added followed by piperidine (200 pL, 2.02 mmoL. 9.6 eq). The mixture was stirred at RT for 1 h. Solvent was evaporated. MeCN (7 mL, 20 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 3 mL, 3 x 9 vol.). It was dried in a vacuum oven at 30 °C overnight. It was isolated 224.2 mg (80 %) of beige solid (11).Fmoc-Phe-Val-Gly-TAG (12)

[0193] FLN-Val-Gly-TAG 11 (171.7 mg, 0.13 mmoL, 1.0 eq) was placed to a r.-b. flask. THF (1 mL, 6 vol.) was added. Fmoc-Phe-OH (100.1 mg. 0.26 mmoL, 2.0 eq) and Oxyma Pure (36.4 mg. 0.26 mmoL, 2.0 eq) were placed to a vial. THF (1 mL, 6 vol.) was added followed by DIC (42 pL, 0.27 mmoL, 2.1 eq). The solution turned yellow. It was let to activate for 2 min and then transferred to the flask with compound 11. The mixture was stirred at RT for 1 h. The solvent was evaporated. MeCN (3 mL, 17 vol.) was added, and the slurry was stirred at RT for 5 min. It was filtered and washed with MeCN (2 x 3 mL, 2 x 17 vol ). It was dried in a vacuum oven at 30 °C overnight. It was isolated 179.6 mg (81 %) of beige solid (12).H2N-Phe-Val-Gly-TAG (13)STEP 1

[0194] Fmoc-Gly-TAG 8 (3.0 g, 2.07 mmoL, 1.0 eq) was placed to a beaker. CH2CI2(10 mL, 3 vol.) was added followed by piperidine (2 mL, 20.67 mmoL, 10.0 eq). The mixture was stirred at RT for 1 h. MeCN (70 mL, 23 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 20 mL. 3 x 7 vol.).STEP 2

[0195] The wet solid from step 1 was placed to a beaker. THF (5 mL, 2 vol.) was added. Fmoc-Val-OH (1.42 g, 4.18 mmoL, 2.0 eq) and Oxyma Pure (604.2 mg, 4.25 mmoL, 2.0 eq) were placed to a vial. THF (5 mL, 2 vol.) was added to the vial followed by DIC (640 pL, 4.13 mmoL, 2.0 eq). The solution turned yellow. It was let to activate for 2 min and then transferred to the beaker. CH2CI2(5 mL, 2 vol.) was added to dissolve remaining solids. The mixture was stirred at RT for 1 h. MeCN (90 mL, 30 vol.) was added, and the slurry was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 20 mL, 3 x 7 vol.).STEP 3

[0196] The wet solid from step 2 was placed to a beaker. CH2CI2(15 mL, 5 vol.) and THF (15 mL, 5 vol.) were added followed by piperidine (2 mL, 20.67 mmoL, 10.0 eq). The mixture was stirred at RT for 1 h. MeCN (180 mL, 60 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 40 mL, 3 x 13 vol.).STEP 4

[0197] The wet solid from step 3 was placed to a beaker. THF (25 mL, 8 vol.) and CH2CI2(15 mL, 5 vol.) were added. Fmoc-Phe-OH (1.62 g, 4.18 mmoL, 2.0 eq) andOxyma Pure (587.3 mg, 4.13 mmoL, 2.0 eq) were placed to a vial. THF (5 mL, 2 vol.) was added to the vial followed by DIC (640 pL, 4.13 mmoL, 2.0 eq). The solution turned yellow. It was let to activate for 2 min and then transferred to the beaker. The mixture was stirred at RT for 1 h. MeCN (250 mL, 80 vol.) was added, and the slurry was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 40 mL, 3 x 13 vol.).STEP 5

[0198] The wet solid from step 4 was placed to a beaker. CH2CI2(30 mL. 10 vol.) and THF (30 mL. 10 vol.) were added followed by piperidine (2 mL, 20.67 mmoL. 10.0 eq). The mixture was stirred at RT for 1 h. MeCN (350 mL, 120 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 40 mL, 3 x 13 vol.). It was dried in a vacuum oven at 30 °C overnight. It was isolated 2. 14 g (70 %) of beige solid.H?N-Phe-Val-Glv-OH (tripeptide 13A)

[0199] H2N-Phe-Val-Gly-TAG 13 (1.0 g, 0.68 mmoL, 1.0 eq) was placed to a r.-b. flask. TFA / H2O 95:5 (10 mL, 10 vol.) was added. Purple slurry was stirred at RT for 1.5 h and turned white during this time. H2O (100 mL, 100 vol.) was added. It was stirred at RT for another 10 min. It was filtered and washed with H2O (3 x 20 mL, 3 x 20 vol ). The aqueous filtrate was extracted with TBME (3 x 50 mL, 3 x 50 vol.). H2O layer was evaporated. TBME (20 mL, 20 vol.) was added. White slurry' was stirred at RT for 10 min. It was filtered and washed with TBME (2 x 5 mL, 2 x 5 vol.). It was dried in a vacuum oven at 30 °C overnight. The tripeptide 13A, H2N-F-V-G-OH, was isolated 166. 1 mg (76 %) of white solid.

[0200] FIG. 6 shows the1H NMR of tripeptide 13A in D2O.1H NMR (400 MHz, D2O): δ = 0.95 (dd, 6H), 2.03 (m, 1H), 3.23 (m, 2H), 3.89 (s, 2H), 4.14 (d, 1H), 4.34 (t, 1H), 7.29 (m, 2H), 7.40 (m, 3H).

[0201] FIG. 7 shows the13C NMR of tripeptide 13A in D2O.13C NMR (100 MHz, D2O): δ = 17.7, 18.2, 30.3, 36.9, 42.0, 54.2, 59.5, 127.9, 129.1, 129.4, 133.6, 168.8. 172.3, 174.2.

[0202] FIG 8 shows the LCMS (ES+) of tripeptide 13A LC-MS m / z: 322.29 (M+H+).

[0203] FIG. 9 shows the LCMS (TIC, ESI) of tripeptide 13A (purity ). LC-MS purity: 99%.B. Synthesis of 6-mer Peptide with TAG 1

[0204] FLN-Phe-Val-Gly-TAG 13 (1.0 g, 0.68 mmoL, 1.0 eq) was placed to a r.-b. flask. THF (8 mL, 8 vol.) was added. Fmoc-Gly-OH (412.0 mg. 1.39 mmoL, 2.0 eq) and Oxyma Pure (194.1 mg, 1.37 mmoL. 2.0 eq) were placed to a vial. THF (2.5 mL. 2.5 vol.) and CH2CI2(2.5 mL, 2.5 vol.) were added followed by DIC (210 pL, 1.36 mmoL, 2.0 eq). The solution turned yellow. It was let to activate for 2 min and then transferred to the flask with compound 13. The mixture was stirred at RT for 1.5 h. The solvent was partially evaporated to app. 5 mL (5 vol.). MeCN (30 mL, 30 vol.) was added, and the slurry was stirred at RT for 10 min. It was filtered and washed with MeCN (3 x 10 mL, 3 x 10 vol.). The wet solid was placed to a beaker. CH2CI2(15 mL, 5 vol.) was added followed by piperidine (670 pL, 6.78 mmoL, 10.0 eq). The mixture was stirred at RT for 1 h. The solvent was partially evaporated to app. 5 mL (5 vol.). MeCN (30 mL, 30 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 10 mL,3 x 10 vol.). It was dried in a vacuum oven at 30 °C overnight. It was isolated 875.9 mg (84 %) of beige solid.H2N-Ile-Gly-Phe-Val-Gly-TAG (17)15

[0205] H2N-Gly-Phe-Val-Gly-TAG 15 (777.8 mg, 0.51 mmoL, 1.0 eq) was placed to a r.- b. flask. THF (5.5 mL, 7 vol.) and CH2C12(5.5 mL, 7 vol.) were added. Fmoc-Ile-OH (362.6 mg, 1.03 mmoL, 2.0 eq) and Oxyma Pure (148.0 mg, 1.04 mmoL. 2.0 eq) were placed to a vial. THF (1.5 mL. 2 vol.) and CH2C12(1.5 mL. 2 vol.) were added to the vial followed by DIG (160 pL, 1.03 mmoL, 2.0 eq). The solution turned yellow. It was let to activate for 2 min and then transferred to the flask with compound 15. The mixture was stirred at RT for 1 h. The solvent was partially evaporated to app. 4 mL (5 vol.). MeCN (30 mL, 39 vol.) was added, and the slurry was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 10 mL, 3 x 13 vol.). The wet solid was placed to a beaker. CH2C12(15 mL, 19 vol.) was added followed by piperidine (500 pL, 5.06 mmoL, 10.0 eq). The mixture was stirred at RT for 1 h. The solvent was partially evaporated to app. 4 mL (5 vol.). MeCN (30 mL, 39 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 10 mL, 3 x 13 vol.). It was dried in a vacuum oven at 30 °C overnight. It was isolated 772.0 mg (92 %) of beige solid (17).H2N-Ala-Ile-Gly-Phe-Val-Gly-TAG (19)

[0206] H2N-Ile-Gly-Phe-Val-Gly-TAG 17 (700.4 mg, 0.43 mmoL, 1.0 eq) was placed to a r.-b. flask. THF (5.5 mL, 8 vol.) and CH2C12(2 mL, 3 vol.) were added. Fmoc-Ala-OH (269.5 mg, 0.87 mmoL, 2.0 eq) and Oxyma Pure (124.5 mg, 0.88 mmoL, 2.0 eq) were placed to a vial. THF (1 mL. 1.5 vol.) and CH2C12(1 mL, 1.5 vol.) were added to the vial followed by DIC (133 pL, 0.86 mmoL, 2.0 eq). The solution turned yellow. It was let to activate for 2 min and then transferred to the flask with compound 17. The mixture was stirred at RT for 1 h. The solvent was partially evaporated to app. 4 mL (6 vol.). MeCN (25 mL, 37 vol.) was added, and the slurry was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 7 mL, 3 x 10 vol.). The wet solid was placed to a beaker. CH2C12(15 mL, 21 vol.) was added followed by piperidine (420 pL, 4.25 mmoL, 9.9 eq). The mixture was stirred at RT for 1 h. The solvent was partially evaporated to app. 4 mL (6 vol.). MeCN (25 mL, 37 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 7 mL, 3 x 10 vol.). It was dried in a vacuum oven at 30 °C overnight. It was isolated 678.3 mg (92 %) of white solid (19).H2N-Ala-Ile-Glv-Phe-Val-Glv-OH (19A)

[0207] H2N-Ala-Ile-Gly-Phe-Val-Gly-TAG 19 (566.4 mg, 0.33 mmoL, 1.0 eq) was placed to a r.-b. flask. TFA / H2O 95:5 (6 mL, 10 vol.) was added. The slurry' was stirred at RT for 2 h. H2O (30 mL, 53 vol.) was added. It was stirred at RT for another 5 min. It was filtered and washed with H2O (3 x 10 mL, 3 x 18 vol.). The aqueous filtrate was extracted with TBME (3 x 25 mL, 3 x 44 vol.). H2O layer was evaporated. TBME (15 mL, 26 vol.) was added. White slurry was stirred at RT for 5 min. It was filtered and washed with TBME (2 x 5 mL, 2 x 9 vol.). It was dried in a vacuum oven at 30 °C overnight. It was isolated 140.6 mg (76 %) of white solid (19A).

[0208] FIG. 10 shows the ’H NMR spectrum of the hexapeptide 19A in D2O. ’H NMR (400 MHz, D2O): δ = 0.91 (m, 12H), 1.24 (m, 1H), 1.53 (d, m, 4H), 1.87 (m, 1H), 2.03 (m, 1H), 3.08 (d, 2H), 3.88 (s, 2H), 3.94 (s, 2H), 4.10 (d, 1H), 4.14 (q, 1H), 4.21 (d, 1H), 4.67 (t, 1H), 7.27 (m, 2H), 7.36 (m, 3H).

[0209] FIG 11 shows the13C NMR spectrum of hexapeptide 19A in D2O.1?C NMR(100 MHz, D2O): δ = 10.1, 14.6. 16.6. 17.5, 18.2, 24.6, 30.3, 36.0, 37.2, 42.1, 48.8, 55.1.58.7, 59.3, 127.2, 128.7, 129.2, 136.0, 170.5, 172.7, 172.8, 173.6, 173.7.

[0210] FIG. 12 shows the LCMS (ES+) chromatograph of hexapeptide 19A. LC-MS m / z: 563.45 (M+H+).

[0211] FIG. 13 shows the LCMS (TIC, ESI) chromatograph of hexapeptide 19A (purity).EC -MS purity: 96 %.C. Synthesis of 10-mer Peptide with TAG 1SchemeH2N-Ser(tBu)-Ala-Arg(Pbf)-Glv-Ala-Ile-Glv-Phe-Val-Glv-TAG (27)STEP 1

[0212] Fmoc-Gly-TAG 8 (5.83 g, 4.01 mmoL, 1.0 eq) was placed to a r.-b. flask. CH2CI2(60 mL, 10 vol.) was added. Piperidine (4.0 mL, 40.49 mmoL, 10.1 eq) was added. The mixture was stirred at RT for 1 h 20 min. Solvent was partially evaporated to 10 mL.MeCN (175 mL, 30 vol.) was added. It was stirred at RT for 10 min. It was filtered and washed with MeCN (3 x 50 mL, 3 x 9 vol.). It was dried in a vacuum oven at 30 °C overnight. It was isolated 4.71 g (95 %) of light-yellow solid (LLN-Gly-TAG 9).STEP 2

[0213] H2N-GIV-TAG 9 (4.61 g, 3.76 mmoL, 1.0 eq) was placed to a r.-b. flask. THF (30 mL, 7 vol.) was added. Fmoc-Val-OH (2.56 g, 7.54 mmoL, 2.0 eq) and Oxyma Pure (1.07 g, 7.54 mmoL, 2.0 eq) were placed to a vial. CH2CI2(10 mL, 2 vol.) and THF (10 mL, 2 vol.) were added to the vial followed by DIG (1.16 mL, 7.51 mmoL, 2.0 eq). The solution turned yellow. It was let to activate for 1 min and then transferred to the flask with compound 9. The mixture was stirred at RT for 1.5 h. The solvent was partially evaporated to 10 mL. MeCN (135 mL, 30 vol.) was added. It was stirred at RT for 10 min. It was filtered and washed with MeCN (3 x 40 mL, 3 x 9 vol.). The wet solid was placed to a beaker. CH2CI2(45 mL, 10 vol.) was added. Piperidine (3.6 mL, 36.44 mmoL, 10.0 eq) was added. The mixture was stirred at RT for 1.5 h. Solvent w as partially evaporated to 10 mL. MeCN (135 mL, 30 vol.) was added. It was stirred at RT for 10 min. It was filtered and washed with MeCN (3 x 30 mL. 3 x 7 vol.). It was dried in a vacuum oven at 30 °C overnight. It was isolated 4.22 g (85 %) of beige solid (H2N-Val-Gly-TAG 11).STEP 3

[0214] H2N-Val-Gly-TAG 11 (4.09 g. 3.08 mmoL, 1.0 eq) was placed to a r.-b. flask.THF (35 mL, 9 vol.) was added. Fmoc-Phe-OH (2.41 g, 6.22 mmoL, 2.0 eq) and Oxyma Pure (877.9 mg, 6.18 mmoL, 2.0 eq) were placed to a vial. CH2CI2(11 mL, 3 vol.) and THF (11 mL. 3 vol.) were added to the vial followed by DIC (950 μL, 6. 14 mmoL. 2.0 eq). The solution turned yellow. It was let to activate for 1 min and then transferred to the flask with compound 11. The mixture was stirred at RT for 1.5 h. The solvent was partially evaporated to 10 mL. MeCN (120 mL, 30 vol.) w as added. It w as stirred at RT for 10 min. It was filtered and washed with MeCN (3 x 30 mL, 3 x 7 vol.). The wet solid was placed to a beaker. CH2CI2(45 mL, 11 vol.) was added. Piperidine (3.0 mL, 30.37 mmoL, 9.9 eq)was added. The mixture was stirred at RT for 1.5 h. Solvent was partially evaporated to 10 mL. MeCN (120 mL, 30 vol.) was added. It was stirred at RT for 10 min. It was filtered and washed with MeCN (3 x 30 mL. 3 x 7 vol.). It was dried in a vacuum oven at 30 °C overnight. It was isolated 3.97 g (87 %) of beige solid (H2N-Phe-Val-Gly-TAG 13).STEP 4

[0215] H2N-Phe-Val-Gly-TAG 13 (3.05 g, 2.07 mmoL, 1.0 eq) was placed to a r.-b. flask. THF (30 mL, 10 vol.) was added. Fmoc-Gly-OH (1.24 g. 4.17 mmoL, 2.0 eq) andOxyma Pure (590.8 mg, 4.16 mmoL, 2.0 eq) were placed to a vial. CH2CI2(8 mL, 3 vol.) and THF (8 mL, 3 vol.) were added to the vial followed by DIC (0.8 mL, 5.17 mmoL, 2.5 eq). The solution turned yellow. It was let to activate for 1 min and then transferred to the flask with compound 13. The mixture was stirred at RT for 1.5 h. The solvent was partially evaporated to 10 mL. MeCN (95 mL, 30 vol.) was added. It w as stirred at RT for 10 min. It was fdtered and washed with MeCN (3 x 30 mL, 3 x 10 vol). It was dried in a vacuum oven at 30 °C overnight. It was isolated 3.46 g (95 %) of beige solid (Fmoc-Gly-Phe-Val- Gly-TAG).STEP S

[0216] Fmoc-Gly-Phe-VaLGly-TAG (3.41 g, 1.94 mmoL, 1.0 eq) was placed to a r.-b. flask. CH2CI2(40 mL, 12 vol.) was added. Piperidine (2.0 mL, 20.25 mmoL, 10.4 eq) was added. The mixture was stirred at RT for 1.5 h. MeCN (180 mL, 50 vol.) was added. It w as stirred at RT for 10 min. It was filtered and washed with MeCN (3 x 50 mL, 3 x 15 vol.). The wet solid was placed to a beaker. THF (25 mL, 7 vol.) and CH2CI2(25 mL, 7 vol.) were added. Fmoc-Ile-OH (1.38 g. 3.90 mmoL, 2.0 eq) and Oxyma Pure (554.7 mg, 3.90 mmoL, 2.0 eq) were placed to a vial. CH2CI2(9 mL, 3 vol.) and THF (9 mL, 3 vol.) were added to the vial followed by DIC (0.6 mL, 3.87 mmoL, 2.0 eq). The solution turned yellow. It was let to activate for 1 min and then transferred to the flask with Fmoc-4mer- TAG. The mixture was stirred at RT for 1.5 h. MeCN (180 mL. 50 vol.) was added. It was stirred at RT for 45 min. It was filtered and washed with MeCN (3 x 50 mL, 3 x 15 vol.). The wet solid was placed to a beaker. CH2CI2(60 mL, 18 vol.) was added. Piperidine (2.0 mL, 20.25 mmoL, 10.4 eq) w as added. The mixture was stirred at RT for 1 h 15 min.MeCN (180 mL, 50 vol.) was added. It was stirred at RT for 10 min. It was filtered and washed with MeCN (3 x 50 mL, 3 x 15 vol.). It was dried in a vacuum oven at RT overnight. It was isolated 2.43 g (76 %) of beige solid (H2N-Ile-Gly-Phe-Val-Gly-TAG17).STEP 6

[0217] I-LN-Ile-Gly-Phe-Val-Gly-TAG 17 (2.31 g, 1.41 mmoL, 1.0 eq) was placed to a r.-b. flask. THF (18 mL, 9 vol.) and CH2CI2(18 mL, 9 vol.) were added. Fmoc-Ala-OH(874.9 mg, 2.81 mmoL, 2.0 eq) and Oxyma Pure (407.3 mg, 2.87 mmoL, 2.0 eq) were placed to a vial. CH2CI2(5 mL, 2 vol.) and THF (5 mL. 2 vol.) were added to the vial followed by DIC (440 pL, 2.84 mmoL, 2.0 eq). The solution turned yellow. It was let to activate for 1 min and then transferred to the flask with Fmoc-5mer-TAG. The mixture wasstirred at RT for 1 h 20 min. MeCN (150 mL, 65 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 45 mL, 3 x 20 vol.). The wet solid was placed to a beaker. CH2CI2(46 mL, 20 vol.) was added. Piperidine (1.4 mL, 14.17 mmoL, 10.0 eq) was added. The mixture was stirred at RT for 1 h 20 min. MeCN (150 mL, 65 vol.) was added. It was stirred at RT for 5 min. It was filtered and w ashed with MeCN (3 x 45 mL, 3 x 20 vol.). The wet solid was placed to a beaker. THF (17 mL, 7 vol.) and CH2CI2(17 mL, 7 vol.) were added. Fmoc-Gly-OH (833.4 mg, 2.80 mmoL, 2.0 eq) and Oxyma Pure (398.0 mg, 2.80 mmoL, 2.0 eq) were placed to a vial. CH2CI2(5 mL, 2 vol.) and THF (5 mL, 2 vol.) were added to the vial followed by DIC (440 pL, 2.84 mmoL, 2.0 eq). The solution turned yellow. It was let to activate for 1 min and then transferred to the flask with Fmoc-6mer-TAG. The mixture was stirred at RT for 1 h 15 min. MeCN (150 mL, 65 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 25 mL, 3 x 11 vol.). The wet solid was placed to a beaker. CH2CI2(46 mL, 20 vol.) was added. Piperidine (1.4 mL, 14.17 mmoL, 10.0 eq) was added. The mixture was stirred at RT for 1 h. MeCN (150 mL, 65 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 30 mL, 3 x 13 vol.). It was dried in a vacuum oven at RT overnight. It was isolated 1.90 g (76 %) of white solid (H2N-Gly-Ala-Ile-Gly-Phe-Val- Gly-TAG).STEP ?

[0218] FLN-Gly-Ala-Ile-Gly-Phe-Val-Gly-TAG (1.82 g, 1.03 mmoL, 1.0 eq) was placed to a r.-b. flask. THF (11 mL, 9 vol.) and CH2CI2(11 mL, 9 vol.) were added. Fmoc- Arg(Pbf)-OH (1.23 g, 1.90 mmoL, 1.8 eq) and Oxyma Pure (269.6 mg, 1.90 mmoL, 1.8 eq) were placed to a vial. THF (20 mL, 11 vol.) was added to the vial. Fmoc-Arg(Pbf)-OH did not fully dissolve. D1C (290 pL. 1.87 mmoL, 1.8 eq) was added. The heterogenous mixture turned yellow. It was let to activate for 1 min and then transferred to the flask with Fmoc-7mer-TAG. The mixture was stirred at RT for 1.5 h. MeCN / DMF 4: 1 (150 mL, 80 vol.) was added. It was stirred at RT for 10 min. It was filtered and washed with MeCN / DMF 4: 1 (2 x 25 mL, 2 x 14 vol.) and MeCN (1 x 25 mL. 1 x 14 vol.). The wet solid was placed to a beaker. CH2CI2(40 mL, 22 vol.) was added. Piperidine (1.0 mL, 10.12 mmoL, 9.8 eq) was added. The mixture was stirred at RT for 1.5 h. MeCN (90 mL, 50 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 25 mL, 3 x 14 vol.). It was dried in a vacuum oven at RT overnight. It was isolated 1.63 g (72 %) of white solid (H2N-Arg(Pbf)-Gly-Ala-Ile-Gly-Phe-Val-Gly-TAG).STEP S

[0219] H2N-Arg(Pbf)-Gly-Ala-Ile-Gly-Phe-Val-Gly-TAG (1.54 g, 0.70 mmoL, 1.0 eq) was placed to a r.-b. flask. THF (12 mL, 8 vol.) and CH2CI2(12 mL, 8 vol.) were added. Fmoc-Ala-OH (439. 1 mg, 1.40 mmoL, 2.0 eq) and Oxyma Pure (202.3 mg, 1.42 mmoL, 2.0 eq) were placed to a vial. CH2CI2(3 mL, 2 vol.) and THF (3 mL, 2 vol.) were added to the vial followed by DIC (220 pL, 1 .42 mmoL, 2.0 eq). The solution turned yellow. It was let to activate for 1 min and then transferred to the flask with Fmoc-8mer-TAG. The mixture was stirred at RT for 1 h. MeCN (100 mL, 65 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 20 mL. 3 x 13 vol.). The wet solid was placed to a beaker. CH2CI2(30 mL, 20 vol.) was added. Piperidine (0.7 mL, 7.09 mmoL, 10.1 eq) was added. The mixture was stirred at RT for 1 h. MeCN (100 mL, 65 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x20 mL, 3 x 13 vol.). The wet solid was placed to a beaker. THF (12 mL, 8 vol.) and CH2CI2(12 mL, 8 vol.) were added. Fmoc-Ser(tBu)-OH (538.9 mg. 1.40 mmoL, 2.0 eq) and Oxyma Pure (198.8 mg, 1.40, 2.0 eq) were placed to a vial. CH2CI2(3 mL. 2 vol.) and THF (3 mL, 2 vol.) were added to the vial followed by DIC (220 pL, 1.42 mmoL, 2.0 eq). The solution turned yellow. It was let to activate for 1 min and then transferred to the flask with Fmoc-9mer-TAG. The mixture was stirred at RT for 1 h. MeCN (100 mL, 65 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 15 mL. 3 x 10 vol ). The wet solid was placed to a beaker. CH2CI2(20 mL, 13 vol.) was added. Piperidine (0.7 mL, 7.09 mmoL, 10.1 eq) was added. The mixture was stirred at RT for 1 h. MeCN (100 mL, 65 vol.) was added. It was stirred at RT for 5 min. It was filtered and washed with MeCN (3 x 20 mL, 3 x 13 vol.). It was dried in a vacuum oven at RT overnight. It was isolated 1.40 g (83 %) of white solid (H2N-Ser(tBu)-Ala-Arg(Pbf)-Gly- Ala-Ile-Gly-Phe-Val-Gly-TAG 27).H2N-Ser-Ala-Arg-Gly-Ala-lle-Gly-Phe-Val-Gly-OH (27A)

[0220] H2N-Ser(tBu)-Ala-Arg(Pbf)-Gly-Ala-Ile-Gly-Phe-Val-Gly-TAG 27 (1.24 g, 0.52 mmoL, 1.0 eq) was placed to a r.-b. flask. TFA / H2O / TIS 95:2.5:2.5 (12.5 mL, 10 vol.) was added. The heterogenous mixture was stirred at RT for 1.5 h. H2O (50 mL, 40 vol.) was added. It was stirred at RT for another 10 min. It was filtered and washed with H2O (3 x 10mL, 3 x 8 vol.). The aqueous filtrate was extracted with TBME (3 x 50 mL, 3 x 40 vol.).H2O layer was evaporated. TBME (20 mL, 16 vol.) was added. White slurry was stirred at RT for 5 min. It was filtered and washed with TBME (3 x 5 mL, 2 x 4 vol.). It was dried in a vacuum oven at 30 °C overnight. It was isolated 152.9 mg (31 %) of white solid.

[0221] Increase of TAG related impurity without peptide chain was observed after extended evaporation of the reaction mixture in the presence of piperidine which possibly contributed to the lower yield. Lower amount of H2O (40 vol.) was used during the cleavage which could potentially result in lower yield as well. Recommendation; do not use evaporation step during the synthesis but direct precipitation with MeCN, use higher amount of H2O (100 vol.) during the cleavage.

[0222] FIG. 14 shows the1H NMR spectrum of decapeptide 27A in D2O.1H NMR (400 MHz, D2O): δ = 0.92 (m, 12H), 1.24 (m, 1H), 1.42 (m, 4H), 1.56 (m, 2H), 1.68 (m, 1H), 1.89 (m, 2H). 2.04 (m, 1H), 3.11 (d, 2H), 3.23 (m. 1H), 3.67 - 4.46 (m, 18H), 4.64 (t, 1H), 7.27 (m, 2H), 7.35 (m, 3H).

[0223] FIG 15 shows the13C NMR spectrum of hexapeptide 27A in D2O.13C NMR (100 MHz, D2O): δ = 10.08. 14.69, 16.50, 17.47, 18.31, 24.27. 24.64, 27.88. 30.26, 35.91.37.10, 42.22, 48.85, 49.48, 54.41, 55.21 , 58.63, 59.24, 60.22, 127.20, 128.76, 129.22,135.98, 162.50 - 175.02.

[0224] FIG. 16 show's the LCMS (ES+) chromatograph of decapeptide 27A. LC-MS m / z: 934.73 (M+H+).

[0225] FIG. 17 show s the LCMS (TIC, ESI) chromatograph of decapeptide 27A (purity).LC-MS purity; 74 %.

[0226] 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

CLAIMS1. A selectively soluble support compound derived from citric acid, the selectively soluble support compound represented by Formula (I):wherein R1is a straight or branched C6-C40alkyl or aliphatic group; R2and R3are each independently selected from H and -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-C30alkyl group, or a C7-C30aralkyl group, wherein the alkyl portion of the C7-C30aralkyl group is a straight or branched C1-C30alkyl.

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

3. The selectively soluble support compound of claim 2, wherein R1is C18H37.

4. The selectively soluble support compound of claim 1, wherein Y is -NHR.

5. The selectively soluble support compound of claim 4. wherein R is a C1-C30alkyl.

6. The selectively soluble support compound of claim 5, wherein R is methyl.

7. The selectively soluble support compound of claim 4, wherein R is H.

8. The selectively soluble support compound of claim 4, wherein R is a C7-C30aralkyl group.

9. 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 (I):wherein R1is a C6-C40straight or branched alkyl or aliphatic group; R2and R3are each independently selected from H and -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-C30alkyl group, or a C7-C30aralkyl group, wherein the alkyl portion of the C7-C30aralkyl group is a straight or branched C1-C30alkyl, 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.

10. The liquid phase method of claim 9, wherein Y is -OH.

11. The liquid phase method of claim 10, wherein R1is C18H3712. 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-phosphonatedform, wherein the selectively soluble support is a compound represented by Formula (I): whereinR1is a straight or branched C6-C40aliphatic group;R.2 and R3are each independently selected from H and -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-C30alkyl group, or a C7-C30aralkyl group, wherein the alkyl portion of the C7-C30aralkyl group is a straight or branched C1-C30alkyl.

13. The liquid phase method of claim 12, further comprising 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.

14. The liquid phase method of claim 13, wherein the elongation reaction cycle comprises: a first step of 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 supportrepresented 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 of converting the resultant 5'-hydroxyl group into an H-phosphonated form using an H-phosphonate reagent, and a third step of forming an oligomer of the first nucleoside or first oligonucleotide with a second nucleoside or second oligonucleotide having a 3'-hydroxyl group and having a 5 '-hydroxyl group protected with a temporary protecting group, by forming a phosphite diester bond from the 5'-hydroxyl group, now converted to the H-phosphonated form, of the first nucleoside or first oligonucleotide and the 3 '-hydroxyl group of the second nucleoside or second oligonucleotide.

15. The liquid phase method of claim 14, further comprising a fourth step of converting the phosphite di ester bond of the oligomer into a phosphodi ester 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.

16. The liquid phase method of claim 14, further comprising a fourth step of converting the phosphite di ester bond of the oligomer into a phosphodiester bond, a thiophosphodiester bond, a boranophosphodiester bond or an aminophosphodiester bond.

17. The liquid phase method of claim 16, 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.

18. The liquid phase method of claim 17, further comprising a step of cleaving the formed oligonucleotide from the compound of Formula (I).

19. The liquid phase method of claim 9, further comprising a step of cleaving the formed peptide from the compound of Formula (I).

20. The selectively soluble support compound of claim 1, wherein one of R2and R3is - O-Methyl.

21. The liquid phase method of claim 9. wherein one of R2and R3is -O-Methyl.

22. The liquid phase method of claim 12, wherein one of R2and R3is -O-Methyl.

23. The selectively soluble support compound of claim 1, wherein R2and R3are not the same.

24. The liquid phase method of claim 9, wherein R2and R3are not the same.

25. The liquid phase method of claim 12, wherein R2and R3are not the same.

26. The selectively soluble support compound of claim 1, wherein the selectively soluble support compound is represented by Formula (II):wherein R1is a straight or branched C6-C40alkyl or aliphatic group; 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-C30alkyl group, or a C7-C30aralkyl group, wherein the alkyl portion of the C7- C30aralkyl group is a straight or branched C1-C30alkyl.

27. The selectively soluble support compound of claim 1, wherein the selectively soluble support compound is represented by Formula (III):wherein R1is a straight or branched Cs-Cro alkyl or aliphatic group; 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-C30alkyl group, or a C7-C30aralkyl group, wherein the alkyl portion of the C7- C30aralkyl group is a straight or branched C1-C30alkyl..

28. The liquid phase method of claim 9, wherein comprises condensing a compound of Formula (II):wherein R1is a C6-C40straight or branched alkyl or aliphatic group; andY is selected from the group consisting of -OH, -COOH, -0-C(0)-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-C30alkyl group, or a C7-C30aralkyl group, wherein the alkyl portion of the C7- C30 aralkyl group is a straight or branched C1-C30alkyl.

29. The liquid phase method of claim 9, wherein comprises condensing a compound of Formula (III): whereinR1is a C6-C40straight or branched alky l or aliphatic group; 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-C30alkyl group, or a C7-C30aralkyl group, wherein the alkyl portion of the C7- C30 aralkyl group is a straight or branched C1-C30alkyl.

30. The liquid phase method of claim 12, wherein the selectively soluble support is a compound represented by Formula (II): whereinR1is a straight or branched C6-C40aliphatic group; 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-C30alkyl group, or a C7-C30aralkyl group, wherein the alkyl portion of the C7- C30 aralkyl group is a straight or branched C1-C30alkyl.

31. The liquid phase method of claim 12, wherein the selectively soluble support is a compound represented by Formula (III); whereinR1is a straight or branched C6-C40aliphatic group; 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-C30alkyl group, or a C7-C30aralkyl group, wherein the alkyl portion of the C7- C30aralkyl group is a straight or branched C1-C30alkyl.

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