Soluble carrier for peptide synthesis and synthesis method using same

Abstract

[Problem] To provide a tag that is for peptide synthesis, that easily dissolves in an organic solvent, that is easy to produce, and that has high functionality. [Solution] An alkoxy-substituted aromatic compound represented by formula (I) (in the formula, X represents (CH2)m, CH(CH3), or C(CH3)2, Y represents a hydroxy group, a thiol group, an amino group, an alkylamino group, a formyl group, or a carboxy group, R's may be the same or different from each other, and each independently represent a linear saturated hydrocarbon group that has 3-30 carbon atoms and that includes at least one heteroatom, n represents an integer of 5 or less, and m represents 0 or a natural number of 1-3).

Description

Soluble support for peptide synthesis and synthesis method using same

[0001] The present invention relates to a novel compound that can be used as a separation carrier. The present invention also relates to a method for synthesizing peptides using the compound.

[0002] There are two main techniques for peptide synthesis: solid-phase peptide synthesis (SPPS) and liquid-phase peptide synthesis (LPPS). Solid-phase peptide synthesis is suitable for small-scale production because purification at each stage of the amino acid elongation reaction is theoretically impossible and the synthesis cost is high. In contrast, liquid-phase peptide synthesis is suitable for mass production, but as the peptide chain lengthens, the peptide elongation reaction becomes more difficult, mainly due to physical properties, making it difficult to synthesize long-chain peptides.

[0003] Therefore, a synthesis method that combines the advantages of the above-mentioned solid-phase peptide synthesis method and solution-phase peptide synthesis method has been investigated (Patent Document 1: JP 2000-44493 A). This synthesis method synthesizes peptides using solution-soluble protecting groups (referring to all protecting groups including those at the C-terminus of amino acids, hereinafter also referred to as "tags"). For example, amino acids are bound to tags having long-chain alkyl groups, and peptides are synthesized by sequentially repeating elongation reactions. According to this synthesis method, only the peptides bound to the tags are solidified at each elongation step, allowing for easy separation and purification of the solidified substances.

[0004] Another known method involves phase separation in a liquid, whereby only peptide components bound to dissolved tags are selectively dissolved in a specific phase, thereby separating them from other unwanted components (hereinafter also referred to as the "tagging liquid-liquid method"). This method allows for separation of unwanted components without solidification, and therefore contributes to speeding up and simplifying the reaction process.

[0005] With the development of liquid-liquid tagging methods, insolubilization of the peptide bound to the tag is no longer a necessary requirement, and highly soluble tags have attracted attention. Tags with improved solubility due to branching of the long-chain alkyl group have been disclosed (Patent Document 2, Non-Patent Document 1, Patent Document 3).

[0006] In addition, a benzyl compound having a group containing an —O—Si— structure at the end of the tag has been disclosed (Patent Document 4). The benzyl compound described in this document is highly soluble in organic solvents and can be effectively used in liquid phase synthesis methods.

[0007] In addition, taking into consideration solubility in organic solvents, a benzyl compound has been disclosed that has one reactive group at the benzyl position and has two or three unsaturated alkoxy groups consisting of unsaturated hydrocarbon groups having 14 to 60 carbon atoms on the benzene ring (Patent Document 5).

[0008] JP 2000-44493, Patent No. 5929756, Patent No. 7260725, Patent No. 6116782, JP 2023-130120

[0009] Daisuke Takahashi et al., Angew. Chem., Int. Ed. 2017, 56, 7803-7807.

[0010] However, the hydrophobic tag in Patent Document 2, which has a branched chain and is considered preferable from the standpoint of solubility, generates stereoisomers due to the branching. Because the tag itself is a mixture of multiple structures, it becomes difficult to evaluate the purity during peptide synthesis. For example, when synthesizing a peptide in a reaction system containing three types of tags, three types of peptides bound to the tags will also be obtained, making it necessary to determine whether the intended product is being obtained, which naturally affects the product yield. Furthermore, the tag in Patent Document 2 requires hydrogenation during the synthesis process, making its production difficult.

[0011] Furthermore, the tag described in Patent Document 3 (Patent No. 7260725) also has a branched structure, which causes the same problems as those in Patent Document 2. Furthermore, separation and purification are difficult.

[0012] Furthermore, in the tag having an —O—Si— structure described in Patent Document 4 (Japanese Patent No. 6116782), the O—Si bond is easily broken, and there is a problem with stability.

[0013] Furthermore, the tag described in Patent Document 5 (JP 2023-130120 A) has a side chain portion with an unsaturated fatty acid structure, which causes stability problems.

[0014] Therefore, there is a need to develop tags that are readily soluble in organic solvents, easy to manufacture, and highly functional.

[0015] In order to solve the above problems, the following inventions are provided.

[0016] A first embodiment of the present invention is a compound of formula (I):

[0017] An alkoxy-substituted aromatic compound represented by formula (I): (wherein X is (CH2)m, CH(CH3), or C(CH3)2; Y is a hydroxy group, a thiol group, an amino group, an alkylamino group, a formyl group, or a carboxy group; R may be the same or different and each independently represents a linear saturated hydrocarbon group having 3 to 30 carbon atoms and containing one or more heteroatoms; n is an integer of 5 or less; and m is 0 or a natural number from 1 to 3.

[0018] The present invention also provides the alkoxy-substituted aromatic compound described above, wherein the heteroatom is oxygen.

[0019] The present invention also provides the above-described alkoxy-substituted aromatic compound, in which the number of heteroatoms is 5 or less.

[0020] The present invention also provides the above-described alkoxy-substituted aromatic compound, wherein m is 1.

[0021] The present invention also provides the above-described alkoxy-substituted aromatic compound, wherein R is selected from the following formulae:

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] The present invention also provides the above-described alkoxy-substituted aromatic compound, wherein the number of heteroatoms is 1 or 2.

[0040] The present invention also provides the above-described alkoxy-substituted aromatic compound, wherein one heteroatom in R is bonded to at least four —CH 2 — groups.

[0041] The present invention also provides a separation support which is the above-described alkoxy-substituted aromatic compound.

[0042] The present invention also provides a reagent for peptide synthesis containing the above-described alkoxy-substituted aromatic compound.

[0043] The present invention also provides an amino acid-added alkoxy-substituted aromatic compound obtained by adding an amino acid to the above-described alkoxy-substituted aromatic compound.

[0044] The present invention also provides a method for separating a protected amino acid or a protected peptide protected on a carrier derived from an unsaturated alkyl-containing aromatic compound represented by formula (I), the method comprising the steps of:

[0045] a step of condensing an alkoxy-substituted aromatic compound represented by formula (I) (wherein X is (CH2)m, CH(CH3), or C(CH3)2, Y is a hydroxy group, a thiol group, an amino group, an alkylamino group, a formyl group, or a carboxy group, and R may be the same or different and each independently represents a straight-chain saturated hydrocarbon group having 3 to 30 carbon atoms and containing one or more heteroatoms, n is an integer of 5 or less, and m is 0 or a natural number of 1 to 3) with an amino acid whose N-terminal amino group is protected ("N-protected") to obtain an N-protected amino acid whose C-terminus is protected with a carrier derived from the alkoxy-substituted aromatic compound ("C-protected"), or a step of condensing an N-protected amino acid with a C-protected amino acid or a C-protected peptide to obtain a C-protected-N-protected peptide; step (2): a step of removing the protecting group at the N-terminus of the carrier-protected-N-protected amino acid or carrier-protected-N-protected peptide obtained in step (1); and Step (3): separating the carrier-protected amino acid or carrier-protected peptide obtained in step (2).

[0046] The present invention also provides the above-described separation method, wherein step (3) is carried out by: (i) a solid-liquid separation step of insolubilizing (crystallizing) the supported amino acid or supported peptide and separating it; or (ii) a liquid separation step of separating an organic solvent containing the supported amino acid or supported peptide from an aqueous solution or organic solvent containing water or an organic solvent containing impurities.

[0047] The present invention also provides the above-described separation method, wherein the N-protected amino acid or N-protected peptide is an amino acid or peptide whose amino group is protected with a t-butoxycarbonyl (Boc) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a benzyloxycarbonyl (Cbz) group, a 2-(trimethylsilyl)ethoxycarbonyl (Teoc) group, or the like.

[0048] The present invention also provides the above-described separation method, wherein the organic solvent or the mixture of organic solvents is at least one organic solvent selected from the group consisting of THF, DMF, pentane, toluene, normal hexane, heptane, cyclohexane, CPME, MTBE, 2-methyl THF, 4-methyl THP, isopropyl acetate, DCM, chloroform, DMSO, 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, methanol, acetonitrile, ethyl acetate, butyl acetate, anisole, and the like, or a mixture of two or more thereof.

[0049] The present invention also provides a method for synthesizing a peptide in a liquid phase, comprising the steps of: Step (A): in an organic solvent or a mixture of organic solvents,

[0050] a step of condensing an amino acid or C-protected peptide having its C-terminus protected on a carrier ("C-protected") derived from an alkoxy-substituted aromatic compound represented by formula (I): (wherein X is (CH2)m, CH(CH3), or C(CH3)2; Y is a hydroxy group, a thiol group, an amino group, an alkylamino group, a formyl group, or a carboxy group; R may be the same or different and each independently represents a straight-chain saturated hydrocarbon group having 3 to 30 carbon atoms and containing one or more heteroatoms; n is an integer of 5 or less; and m is a natural number of 0 or 1 to 3), with an amino acid or N-protected peptide having its N-terminal amino group protected ("N-protected"), in the presence of a condensing agent, to obtain a C-protected-N-protected peptide; step (B): a step of deprotecting the N-terminal protecting group of the C-protected-N-protected peptide to obtain a C-protected peptide; and step (C): a step of separating the C-protected peptide. Also provided is a method for synthesizing a peptide, comprising:

[0051] The present invention also provides the above-described peptide synthesis method, wherein the N-protected amino acid or N-protected peptide is an amino acid or N-Fmoc-protected peptide in which the amino group is protected with a t-butoxycarbonyl group (Boc group), a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a benzyloxycarbonyl (Cbz) group, a 2-(trimethylsilyl)ethoxycarbonyl (Teoc) group, or the like ("N-Fmoc-protected").

[0052] The present invention also provides the aforementioned method for peptide synthesis, wherein the organic solvent or mixture of organic solvents is at least one organic solvent selected from the group consisting of THF, DMF, pentane, toluene, normal hexane, heptane, cyclohexane, CPME, MTBE, 2-methyl THF, 4-methyl THP, isopropyl acetate, DCM, chloroform, DMSO, 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, methanol, acetonitrile, ethyl acetate, butyl acetate, and anisole, or a mixture of two or more thereof.

[0053] The present invention also provides the above-described peptide synthesis method, which further comprises, after the step (A), the following step (a1): adding a water-soluble amine to the reaction solution after the condensation reaction.

[0054] The present invention also provides the above-described method for synthesizing a peptide, wherein the step (B) is a step of carrying out deprotection in the presence of a water-soluble amine.

[0055] The present invention also provides the aforementioned peptide synthesis method, wherein step (C) is a step of neutralizing the reaction solution by adding an acid, washing with an acidic aqueous solution, separating the reaction solution, removing the aqueous layer, obtaining an organic layer, and isolating the C-carrier-protected peptide.

[0056] The present invention also provides the above-described peptide synthesis method, which comprises repeating the above steps one or more times using the C-carrier protected peptide separated in step (C).

[0057] The present invention also provides use of the above-described alkoxy-substituted aromatic compound for peptide synthesis and / or separation of a carrier-protected amino acid or a carrier-protected peptide.

[0058] The present invention provides a novel separation support. In one embodiment of the present invention, the saturated alkyl-containing aromatic compound of the present invention is used as a support in liquid-phase peptide synthesis, enabling highly efficient peptide synthesis. Furthermore, the saturated alkyl-containing aromatic compound of the present invention is easier to produce and more stable than conventional peptide synthesis tags. Furthermore, since the saturated alkyl-containing aromatic compound of the present invention is soluble in a wider range of commonly used organic solvents than conventional peptide synthesis tags, organic solvents with a lower environmental impact can be used in liquid-phase peptide synthesis. Due to its high solubility, the amount of organic solvent used can be reduced, i.e., the peptide production efficiency per unit volume can be improved.

[0059] FIG. 1 shows a chromatogram of protected leucine enkephalin (compound 47) synthesized in Example 20. FIG. 2 shows a chromatogram of leucine enkephalin (compound 48) synthesized in Example 21. FIG. 3 shows a chromatogram of protected leucine enkephalin (compound 51) synthesized in Example 29. FIG. 4 shows a chromatogram of leucine enkephalin (compound 48) synthesized in Example 30. FIG. 5 shows a chromatogram of protected leucine enkephalin (compound 53) synthesized in Example 32. FIG. 6 shows a chromatogram of leucine enkephalin (compound 48) synthesized in Example 33. FIG. 7 shows a chromatogram of protected neuprorelin (compound 55) synthesized in Example 35. FIG. 8 shows a chromatogram of neuprorelin (compound 56) synthesized in Example 36. Figure 9 shows a chromatogram of the protected neurprorelin (compound 58) synthesized in Example 38. Figure 10 shows a chromatogram of the neurprorelin (compound 56) synthesized in Example 39. Figure 11 shows a chromatogram of the protected neurprorelin (compound 60) synthesized in Example 41. Figure 12 shows a chromatogram of the neurprorelin (compound 56) synthesized in Example 42. Figure 13 shows a chromatogram of Fmoc-Ala-Ala-Ala-Ala-O-TagA (compound 62) synthesized in Example 44. Figure 14 shows a chromatogram of Fmoc-Ala-Ala-Ala-Ala-O-TagB (compound 64) synthesized in Example 46. Figure 15 shows a chromatogram of Fmoc-Ala-Ala-Ala-Ala-O-TagC (compound 66) synthesized in Example 48. Figure 16 shows a chromatogram of Fmoc-Ala-Ala-Ala-Ala-O-TagE (Compound 68) synthesized in Example 50. Figure 17 shows the results of a comparative experiment on the condensation reaction between the compound of the present invention synthesized in Example 51 and a conventional tag.

[0060] The present invention will be described below, taking exemplary embodiments as examples, along with preferred methods and materials that can be used in carrying out the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Furthermore, any materials and methods equivalent or similar to those described herein can be similarly used in carrying out the present invention. Furthermore, all publications and patents cited in this specification in connection with the inventions described herein are incorporated by reference, for example, to describe methods, materials, and the like that can be used in the present invention.

[0061] As used herein, the expression "X to Y" means that X is the lower limit and Y is the upper limit, or X is the upper limit and Y is the lower limit. As used herein, "about" is used to mean that a tolerance of ±10% is permitted. As used herein, the term "amino acid" includes natural amino acids, unnatural amino acids, L-amino acids, and D-amino acids, as well as unusual amino acids such as amino acid amides. Furthermore, it also includes amino acids that have been modified in any way that can be prepared with reference to known techniques.

[0062] [Novel Peptide Synthesis Tag] The saturated alkyl-containing aromatic compound of the present invention (hereinafter referred to as "the compound of the present invention") is a compound represented by the following formula (I).

[0063] Formula (I) The symbols in the formula are defined as follows: X is (CH2)m, CH(CH3), or C(CH3)2, Y is a hydroxy group, a thiol group, an amino group, an alkylamino group, a formyl group, or a carboxy group, R may be the same or different and each independently represents a linear saturated hydrocarbon group having 3 to 30 carbon atoms and containing one or more heteroatoms, n is an integer of 5 or less, and m is 0 or a natural number from 1 to 3.

[0064] The compound of the present invention has one reactive group Y and five or less linear saturated hydrocarbon groups having 3 to 30 carbon atoms on the benzene ring. The reactive group Y is a hydroxyl group, a thiol group, an amino group, an alkylamino group, a formyl group, or a carboxyl group, preferably a hydroxyl group, an amino group, or an alkylamino group, and more preferably a hydroxyl group. Specific examples of the reactive group Y include OH, NH, NHCH, and NHCH.

[0065] In the compound of the present invention, the number of carbon atoms in the linear saturated hydrocarbon group is 3 to 30, more preferably 6 to 25, even more preferably 12 to 22, and even more preferably 18 to 20. The five or less linear saturated hydrocarbon groups may be the same or different, but are preferably the same. In addition, there are no particular limitations on the position of the one or more heteroatoms contained in the five or less linear saturated hydrocarbon groups.

[0066] In the compounds of the present invention, one or more heteroatoms in the linear saturated hydrocarbon group are preferably selected from nitrogen, oxygen, silicon, phosphorus, sulfur, fluorine, bromine, chlorine, iodine, etc., with oxygen being particularly preferred from the viewpoints of compound stability and ease of preparation. Furthermore, in the compounds of the present invention, the number of heteroatoms contained in each R is preferably one or more, preferably five, preferably four, preferably three, and preferably two. According to the findings of the present inventors, heteroatoms are thought to affect physical properties by interrupting the continuous carbon chain. In the compounds of the present invention, each linear saturated hydrocarbon group preferably has two or three, preferably four or five, preferably six or seven, preferably eight or nine, preferably ten or eleven, preferably twelve or thirteen, and preferably four or fifteen -CH2- groups connected via the heteroatoms.

[0067] In the compound of the present invention, n in formula (I) is an integer of 5 or less, preferably 4, preferably 3, and preferably 2. The position of the straight-chain saturated hydrocarbon group on the benzene ring in the compound of the present invention is not particularly limited, but suitable examples include 2,4-substituted, 2,5-substituted, 2,6-substituted, 3,4-substituted, 3,5-substituted, 2,3,4-substituted, 2,4,5-substituted, 2,4,6-substituted, or 3,4,5-substituted, preferably 2,4-substituted, 2,5-substituted, 3,5-substituted, 2,4,5-substituted, or 3,4,5-substituted, and more preferably 2,4-substituted, 3,5-substituted, or 3,4,5-substituted.

[0068] Examples of the linear saturated hydrocarbon group on the benzene ring in the compound of the present invention, when the heteroatom is oxygen, can be composed of any one of the following or a combination thereof:

[0069] R in the compounds of the present invention is preferably selected from the following formulae:

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] [Synthesis of the tag of the present invention] The compound of the present invention can be produced by appropriately referring to known methods. When m in X is 1 and Y is a hydroxy group, or when m in X is 0 and Y is a carboxy group, the compound can be produced, for example, by the following steps, but is not limited thereto.

[0088]

[0089] In the above diagram, a saturated alkyl bromide, which serves as the linear saturated hydrocarbon group ("R" in formula (I)) of the compound of the present invention, is produced (R-Br) using the corresponding dibromoalkane, alcohol, or the like as a starting material, by appropriately referring to known methods. Using this, a benzaldehyde or methyl benzoate having n hydroxy groups is alkylated to obtain a saturated alkoxybenzaldehyde or saturated alkoxymethyl benzoate in which the hydrogen atoms of the hydroxy groups are substituted with saturated alkyl groups. The compound of the present invention of formula (I) in which m in X is 1 and Y is a hydroxy group, or the compound of the present invention of formula (I) in which m in X is 0 and Y is a carboxy group, can be obtained by reducing the resulting benzaldehyde or methyl benzoate, or by hydrolyzing the saturated methyl benzoate.

[0090] In the above diagram, alkylation of benzaldehyde or methyl benzoate having n hydroxy groups is carried out using the corresponding alkyl bromide (R-Br) by Williamson ether synthesis in the presence of, but not limited to, K2CO3 in DMF. The resulting benzaldehyde or methyl benzoate can be reduced with, but not limited to, LiAlH4 in THF to give the compound of the present invention of formula (I) in which m in X is 1 and Y is a hydroxy group. The resulting methyl benzoate can be hydrolyzed with, but not limited to, LiOH in THF to give the compound of the present invention of formula (I) in which m in X is 0 and Y is a carboxy group.

[0091] R-Br can be commercially available, or, for example, if it corresponds to 2 in Chemical Formula 2, it can be synthesized separately as shown in the following formula.

[0092]

[0093] In the above diagram, dibromooctane and nonanol are subjected to, but not limited to, Williamson ether synthesis in the presence of NaH in THF to obtain the compound corresponding to R shown in Chemical Formula 2.

[0094] The compounds of the present invention have high solubility in organic solvents due to their long-chain linear alkyl groups, which is expected to result in excellent reactivity. Furthermore, when the alkyl chain is interrupted by a heteroatom, they remain highly stable even after binding to an amino acid, making them suitable for use in peptide synthesis. R-Br and the compounds of the present invention can be produced as single isomers by the widely used Williamson ether synthesis, making their synthesis, separation, and purification easy. Neither the existence nor the effectiveness of compounds with these characteristics that can be used as tags for peptide synthesis have been proven.

[0095] As mentioned above, the properties of alkyl chains in soluble tags have been extensively explored in this field. For efficient separation of tag-bound peptides from highly polar reagents such as amino acids and condensation agents, tags are preferably hydrophobic, and alkyl chains of a certain length are generally preferred. However, long alkyl chains, which are required for hydrophobicity, tend to form ordered structures and aggregate in the liquid phase. Since aggregation is not necessary in the liquid-liquid tagging method, which does not necessarily require solidification, tags with branched or complex side chains have been attracting attention for this purpose. However, as mentioned above, these tags have certain disadvantages in peptide synthesis. In response to this issue, incorporating heteroatoms that disrupt the order of the linear alkyl chains has enabled the creation of highly soluble tags for peptide synthesis while maintaining side chain stability.

[0096] As used herein, the term "organic solvent" refers to a solvent commonly used in this technical field, such as a solvent used in liquid-phase peptide synthesis. Non-limiting examples include THF, DMF, pentane, toluene, normal hexane, heptane, cyclohexane, CPME, MTBE, 2-methyl THF, 4-methyl THP, isopropyl acetate, chloroform, dichloromethane (DCM), dimethyl sulfoxide (DMSO), 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, ethyl acetate, butyl acetate, anisole, and a mixture of two or more of these solvents. The saturated solubility of the compound of the present invention in 100 g of THF at 20°C can be, for example, 1 to 95% by weight.

[0097] The solubility of the compound of the present invention in an organic solvent is shown below. The lower limit of the saturated solubility of the compound of the present invention in 100 g of the organic solvent at 20° C. is not particularly limited as long as the compound binds to the reaction substrate and the subsequent reaction proceeds, but from the viewpoint of industrially stably proceeding with any substrate, the solubility is preferably 2% by weight, more preferably 5% by weight, even more preferably 10% by weight, even more preferably 25% by weight, and particularly preferably 50% by weight.

[0098] The compounds of the present invention can be bound to amino acids or peptides and then separated and recovered together with the amino acids or peptides. Separation and recovery can be easily achieved by insolubilizing the amino acids or peptides bound to the compounds of the present invention that are dissolved in a solvent. The insolubilization conditions can be determined by referring to known reports, such as WO2007 / 034812 and WO2007 / 122847. The compounds of the present invention are also readily soluble in solvents that are easy to separate, such as cyclohexane, 2-methyl THF, isopropyl acetate, CPME, DCM, toluene, ethyl acetate, butyl acetate, and anisole. Therefore, the amino acids or peptides bound to the compounds of the present invention can be easily separated from the reaction residue by a separation procedure. The separation procedure can be performed, for example, by adding water, a mixture of water and a hydrophilic organic solvent, or an organic solvent (e.g., a polar organic solvent) immiscible with the organic solvent in which the compounds of the present invention are dissolved, to a reaction solution containing the amino acids or peptides bound to the compounds of the present invention, stirring and washing, and removing the reaction residues soluble in water and / or the hydrophilic organic solvent by layer separation. Therefore, the compound of the present invention can also be used as a carrier for separating amino acids and peptides. The present invention provides a carrier for separation, and also includes the use of the compound of the present invention as a carrier for separation. Furthermore, the present invention provides a separation method using the compound of the present invention.

[0099] [Method of Peptide Synthesis] The compound of the present invention can be used in peptide synthesis as a protecting group or carrier for the C-terminus or N-terminus of an amino acid or peptide, or for a side chain functional group of an amino acid. Thus, the present invention provides a peptide synthesis technique and also includes the use of the compound of the present invention as a protecting group. Furthermore, the present invention also includes a method of peptide synthesis using the compound of the present invention.

[0100] In the separation methods and peptide synthesis methods using the compounds of the present invention, commonly known amino acids and peptides can be used without limitation. The amino acids may be natural or unnatural amino acids, either L- or D-amino acids, or unusual amino acids such as amino acid amides. Unnatural amino acids are not particularly limited and can include any known unnatural amino acids, including, but not limited to, N-methyl-modified amino acids, N-2,4-dimethoxybenzyl-modified amino acids, 2-aminoisobutyric acid, D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and L-octahydroindoline-2-carboxylic acid. Furthermore, amino acids protected with protecting groups that temporarily protect the side chain functional groups of natural, unnatural, or unusual amino acids, as needed, can also be used in the methods of the present invention. Furthermore, natural, unnatural, or unusual amino acids that have been optionally modified in accordance with known techniques can also be used in the methods of the present invention. The optional modifications are not particularly limited, and any modifications that can be added to amino acids using known or conventional techniques in the technical field of amino acid synthesis can be used without limitation. Examples of such modifications include addition of low molecular weight organic compounds, phosphorylation, biotinylation, PEGylation, glycosylation, and fluorescent modification. Peptides may be composed of any combination of the above amino acids, and in addition, pseudoproline-type dipeptides and O-acylisopeptides can also be used in the present invention.

[0101] The peptide synthesis method using the compound of the present invention can be carried out by the following steps: Step (A): a step of condensing an amino acid or C-protected peptide whose C-terminus has been protected with a support derived from the compound of the present invention (hereinafter referred to as "C-protected") with an amino acid or N-protected peptide whose N-terminal amino group has been protected (hereinafter referred to as "N-protected") in the presence of a condensing agent in an organic solvent or a mixture of organic solvents to obtain a C-protected-N-protected peptide (hereinafter sometimes referred to as the condensation reaction step of the present invention); Step (B): a step of deprotecting the N-terminal protecting group of the C-protected-N-protected peptide to obtain a C-protected peptide (hereinafter sometimes referred to as the deprotection step of the present invention); and Step (C): a step of separating the C-protected peptide (hereinafter sometimes referred to as the separation / recovery step of the present invention).

[0102] The peptide synthesis method using the compound of the present invention can further include, after step (A), step (a1): a step of adding a water-soluble amine, a water-soluble thiol, or the like to the reaction solution after the condensation reaction (hereinafter, this may be referred to as the scavenging reaction step of the present invention).

[0103] In the method for synthesizing a peptide using the compound of the present invention, step (B) can be a step of carrying out deprotection in the presence of a water-soluble amine, a water-soluble thiol, or the like.

[0104] In the method for synthesizing amino acids or peptides using the compound of the present invention, step (C) can be a step of neutralizing the reaction solution by adding an acid, washing with an acidic aqueous solution, separating the reaction solution, removing the aqueous layer, obtaining an organic layer, and isolating the C-carrier protected peptide (hereinafter, sometimes referred to as the acidic aqueous solution washing step of the present invention).

[0105] In the method for synthesizing amino acids or peptides using the compound of the present invention, steps (A) to (C) can be repeated one or more times using the C-carrier protected peptide obtained in step (C).

[0106] The N-terminus of the N-protected amino acid (or peptide) in step (A) of the peptide synthesis method of the present invention can be protected using a known protecting group, such as a t-butoxycarbonyl (Boc) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a benzyloxycarbonyl (Cbz) group, or a 2-(trimethylsilyl)ethoxycarbonyl (Teoc) group, with the Fmoc group being preferred. The peptide synthesis method of the present invention will be described below with reference to an example using an N-Fmoc-protected amino acid (or peptide).

[0107] 1. Solvents Solvents that can be used in the peptide synthesis method of the present invention are not particularly limited and include solvents used in liquid-phase peptide synthesis. Examples of solvents include, but are not limited to, THF, DMF, pentane, toluene, normal hexane, heptane, cyclohexane, CPME, MTBE, 2-methyl THF, 4-methyl THP, isopropyl acetate, chloroform, DCM, DMSO, 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, ethyl acetate, butyl acetate, and anisole. Preferred examples include THF, DMF, toluene, cyclohexane, CPME, MTBE, 2-methyl THF, 4-methyl THP, isopropyl acetate, DMSO, N-methylpyrrolidone, ethyl acetate, butyl acetate, and anisole. Furthermore, a mixture of two or more of the above solvents may also be used.

[0108] A polar solvent is used to insolubilize a peptide bound to the compound of the present invention (carrier) for preparing a starting material for use in the peptide synthesis method of the present invention or for recovering a peptide synthesized using the peptide synthesis method of the present invention. Examples of polar solvents that can be used include methanol, ethanol, isopropanol, acetonitrile, propionitrile, DMF, dimethylacetamide, dimethyl sulfoxide, water, and mixtures of two or more of these. Among these, methanol or acetonitrile is preferably used.

[0109] 2. Carrier for liquid phase peptide synthesis The carrier used in the peptide synthesis method of the present invention is a compound derived from an alkoxy-substituted aromatic compound represented by the following formula (I):

[0110] Formula (I) The symbols in the formula are defined as follows: X is (CH2)m, CH(CH3), or C(CH3)2, Y is a hydroxy group, a thiol group, an amino group, an alkylamino group, a formyl group, or a carboxy group, R may be the same or different and each independently represents a linear saturated hydrocarbon group having 3 to 30 carbon atoms and containing one or more heteroatoms, n is an integer of 5 or less, and m is 0 or a natural number from 1 to 3.

[0111] The compound of the present invention is preferably a compound that has the property of reversibly changing between a dissolved state and an insoluble state depending on a change in the composition of the solvent in which it is dissolved. In an embodiment of the peptide synthesis method of the present invention, the compound of the present invention, which serves as a carrier, can be insolubilized and separated, but it is not essential to insolubilize the carrier, nor is it essential to change the composition of the solvent so as to insolubilize the carrier. Examples of the step of insolubilizing the carrier include, but are not limited to, a step of preparing a C-carrier-protected amino acid (or peptide) to be used in the peptide synthesis method of the present invention, and a step of recovering a C-carrier-protected peptide dissolved in the organic layer (organic solvent or organic solvent mixture layer) obtained by the peptide synthesis method of the present invention.

[0112] 3. Linker: The C-carrier-protected amino acid (or peptide) used in the peptide synthesis method of the present invention is bound to the carboxy group of the amino acid or peptide, either directly or via a linker, to the compound of the present invention. The linker referred to here is an organic compound having two reactive groups, one of which binds to the carboxy group of the amino acid or peptide and the other of which binds to reactive group Y of the compound of the present invention, which serves as the carrier. Preferably, the linker that can be used in the present invention is an organic compound having a molecular weight of about 2000 or less (preferably about 1500 or less, more preferably about 1000 or less) and having at least two reactive groups, which may be the same or different, selected from the group consisting of amino groups, carboxyl groups, and halomethyl groups, within the molecule. Examples of such reactive groups include, but are not limited to, the following compounds:

[0113]

[0114] (wherein n is an integer of 1 to 6, preferably 1 to 4).

[0115] wherein X is a halogen atom, preferably chlorine or bromine.

[0116] (wherein n is an integer of 2 to 40, preferably 2 to 35, more preferably 2 to 28.) (The structural formula of the linker above shows the state before it is bonded to the carboxyl group of the amino acid or peptide and before it is bonded to the reactive group of the compound of the present invention, which is the carrier.)

[0117] In preparing the C-carrier-protected amino acid (or peptide) containing the linker, the order of attachment to the linker is not particularly limited. One of the linkers may be attached to the amino acid or peptide, and then the other may be attached to the compound of the present invention as a carrier. Alternatively, one of the linkers may be attached to the compound of the present invention as a carrier, and then the other may be attached to the amino acid or peptide. Attachment of one of the linkers to the amino acid or peptide can be carried out by appropriately referring to known methods depending on the linker group and the amino acid or peptide group to be attached. Examples include, but are not limited to, amidation using DIPCI / HOBt. Attachment of one of the linkers to the compound of the present invention as a carrier can be carried out by appropriately referring to known methods depending on the linker group and the carrier group to be attached. Examples include, but are not limited to, esterification using DIPCI.

[0118] 4. N-Fmoc-protected amino acids and N-Fmoc-protected peptides An amino acid whose amino group is protected with a 9-fluorenylmethyloxycarbonyl (Fmoc) group (N-Fmoc-protected amino acid) or an N-Fmoc-protected peptide refers to an amino acid or peptide in which the amino group is protected with an Fmoc group, while the carboxyl group is unprotected and reactive. When an amino acid or peptide has one or more amino groups, it is sufficient that at least one amino group is protected with an Fmoc group.

[0119] When the N-Fmoc-protected amino acid (or peptide) has a highly reactive functional group such as a hydroxyl group, an amino group, a guanidyl group, a carboxyl group, a thiol group, an indole group, or an imidazole group, a general protecting group used in peptide synthesis may be introduced into this functional group, and the target compound can be obtained by removing the protecting group as necessary at any time point after completion of the reaction.

[0120] Examples of protecting groups for hydroxyl groups include tBu, Trt, Bz, acetyl, and silyl groups; examples of protecting groups for amino groups include Boc, Fmoc, Cbz, Trt, Mmt, and ivDde groups; examples of protecting groups for guanidyl groups include Pbf, Pmc, and nitro groups; examples of protecting groups for carboxyl groups include tBu, methyl, ethyl, and Bz groups; examples of protecting groups for thiol groups include Trt, Acm, tBu, and S-tBu groups; examples of protecting groups for indole groups include Boc groups; and examples of protecting groups for imidazole groups include Boc, Bom, Bum, and Trt groups.

[0121] Other examples include peptides with a pseudoproline structure in which a hydroxyl or thiol group is acetal-bridged with an α-amino group and protected with Fmoc, and O-acylisopeptides in which an N-Fmoc-protected amino acid is ester-linked to a hydroxyl group and the α-amino group is protected with a Boc group.

[0122] 5. C-Carrier-Protected Amino Acids and C-Carrier-Protected Amino Acid Peptides: An amino acid (C-carrier-protected amino acid) or C-carrier-protected peptide protected on a support for liquid phase peptide synthesis derived from the compound of the present invention refers to an amino acid or peptide in which the C-terminal carboxyl group is protected by the compound of the present invention (support) directly or via a linker, and at least the N-terminal amino group is reactive. When the C-carrier-protected amino acid (or peptide) has highly reactive functional groups such as a hydroxyl group, amino group, guanidyl group, carboxyl group, thiol group, indole group, or imidazole group, these functional groups may be introduced with common protecting groups used in peptide synthesis, and the target compound can be obtained by removing the protecting group, if necessary, after completion of the reaction.

[0123] Examples of protecting groups for hydroxyl groups include tBu, Trt, Bz, acetyl, and silyl groups; examples of protecting groups for amino groups include Boc, Fmoc, Cbz, Trt, Mmt, and ivDde groups; examples of protecting groups for guanidyl groups include Pbf, Pmc, and nitro groups; examples of protecting groups for carboxyl groups include tBu, methyl, ethyl, and Bz groups; examples of protecting groups for thiol groups include Trt, Acm, tBu, and S-tBu groups; examples of protecting groups for indole groups include Boc groups; and examples of protecting groups for imidazole groups include Boc, Bom, Bum, and Trt groups.

[0124] 6. Peptide Synthesis Method The peptide synthesis method of the present invention may involve solid-liquid separation between each of the steps of the condensation reaction, the Fmoc group deprotection, and the C-carrier protected peptide separation and recovery, or these steps may be carried out continuously without solid-liquid separation. Preferably, however, these steps are carried out continuously without solid-liquid separation. Furthermore, in the peptide synthesis method of the present invention, impurities generated during the synthesis process can be reduced or removed by liquid separation. Therefore, the method is suitable as a continuous peptide synthesis method.

[0125] A preferred embodiment of peptide synthesis by the peptide synthesis method of the present invention comprises the following steps (i) to (iv), which include an optional scavenging reaction step and / or an acidic aqueous solution washing step. The following describes the case where Fmoc is used as the amino-protecting group, but the same procedure can also be carried out using Boc as the protecting group. (i) condensation reaction step: a step of condensing a C-protected amino acid or a C-protected peptide with an N-Fmoc-protected amino acid or an N-Fmoc-protected peptide in the presence of a condensing agent in an organic solvent or a mixture of organic solvents to obtain a C-protected-N-Fmoc-protected peptide; (ii) scavenging reaction step: a step of adding a water-soluble amine (hereinafter, sometimes referred to as an amine scavenger) to the reaction solution after the condensation reaction to form a scavenged amino acid activated ester; (iii) Fmoc deprotection step: a step of deprotecting the Fmoc group from the protected N-terminus in the presence of a water-soluble amine; and (iv) acidic aqueous solution washing step: a step of adding an acid to neutralize the reaction solution, further adding an acidic aqueous solution to wash, followed by separation, removing the aqueous layer, and recovering the organic solvent layer containing the C-protected peptide.

[0126] The above steps do not require solid-liquid separation procedures involving insolubilization of the C-protected amino acid, C-protected peptide, or C-protected-N-Fmoc-protected peptide, and can therefore be carried out continuously. Furthermore, by carrying out the above steps, the C-protected peptide to which the amino acid or peptide has been added can be recovered in a dissolved state in the organic layer, compared to the state at the start of the synthesis reaction. Furthermore, since impurities have been reduced or removed from the organic layer in which the C-protected peptide has been dissolved, the next peptide synthesis reaction can be carried out in this state. Thus, one embodiment of the peptide synthesis method using the compound of the present invention can be a peptide synthesis method comprising repeating the above steps (i) to (iv) as many times as necessary.

[0127] The C-protected amino acid or C-protected peptide used in step (i) of the peptide synthesis method of the present invention can be prepared by appropriately referring to known methods used in peptide synthesis. The following describes the C-protected amino acid as an example. For example, the compound of the present invention (support) can be bound to the carboxyl group of an amino acid directly or via a linker. For example, without limitation, a support compound can be dissolved in a solvent such as THF, and an N-Fmoc-protected amino acid and a condensing agent, such as DIPCI, are added to perform condensation to prepare a C-protected-N-Fmoc-protected amino acid, which is an intermediate in which a support is bound to the carboxyl group of the amino acid. Alternatively, a support compound can be dissolved in a solvent such as THF, and a Boc-protected amino acid and a condensing agent, such as DIPCI, are added to perform condensation to prepare a C-protected-N-Boc-protected amino acid, which is an intermediate in which a support is bound to the carboxyl group of the amino acid.

[0128] The prepared C-protected-N-Fmoc-protected amino acid or C-protected-N-Boc-protected amino acid can be obtained with high purity, preferably by insolubilization and recovery. For example, but not limited to, the reaction solution containing the C-protected-N-Fmoc (or Boc)-protected amino acid is distilled under reduced pressure, and then a solvent that insolubilizes the C-protected-N-Fmoc (or Boc)-protected amino acid, such as methanol or acetonitrile, is added to the residue to cause precipitation. The precipitate is filtered and then washed by suspension in a solvent, and the resulting solid is dried to obtain the C-protected-N-Fmoc (or Boc)-protected amino acid.

[0129] The N-terminal protecting group of the C-protected N-Fmoc (or Boc) protected amino acid thus obtained can be removed by appropriate reference to known methods used in peptide synthesis to prepare a C-protected amino acid. For example, but not limited to, the C-protected N-Fmoc protected amino acid can be dissolved in a solvent such as THF, and an Fmoc deprotection reagent such as DBU or piperazine can be added to carry out the Fmoc deprotection reaction. Alternatively, the C-protected N-Boc protected amino acid can be dissolved in a solvent such as CPME, and an Fmoc deprotection reagent such as TFA can be added to carry out the Boc deprotection reaction.

[0130] The C-protected amino acid thus obtained can be prepared in a solution state, and can be subjected to the same acidic aqueous solution washing step as in step (iv) of the present invention. The C-protected amino acid after washing with the acidic aqueous solution can be used in the method of the present invention as the starting material for step (i) of the present invention. In such a case, continuous peptide synthesis is possible, including the step of preparing the C-protected amino acid.

[0131] Furthermore, if necessary, the obtained C-protected amino acid can be recovered by insolubilization. For example, but not limited to, the reaction solution containing the C-protected amino acid can be distilled under reduced pressure, and then a solvent that insolubilizes the support, such as methanol or acetonitrile, can be added to the residue to cause precipitation. The precipitate is filtered and then washed by suspension in a solvent, and the resulting solid can be dried to obtain the C-protected amino acid.

[0132] The C-protected amino acid thus obtained can be used as a starting material in the peptide synthesis method of the present invention. Thus, one embodiment of the peptide synthesis method of the present invention is a peptide synthesis method further comprising a step of preparing a C-protected amino acid prior to the above steps (i) to (iv).

[0133] The C-protected peptide obtained using the peptide synthesis method of the present invention can be recovered using known methods used in the field of peptide synthesis. For example, it can be recovered from the solvent by crystallization. For example, but not limited to, the organic layer containing the obtained C-protected peptide is distilled off under reduced pressure, and then a poor solvent, such as cold acetonitrile, is added to the residue to cause precipitation. The precipitate is filtered and then suspended and washed with a solvent. The resulting solid is dried to obtain the synthesized C-protected peptide. Therefore, one embodiment of the peptide synthesis method of the present invention includes a step of crystallizing and separating the C-protected peptide after the above steps (i) to (iv).

[0134] Each step will be described below. In the following description, a C-protected peptide and an N-Fmoc-protected amino acid are described as examples, but the C-protected peptide is an example of a C-protected amino acid or a C-protected peptide that can be used in the present invention, and the N-Fmoc-protected amino acid is an example of an N-Fmoc-protected amino acid or an N-Fmoc-protected peptide that can be used in the present invention.

[0135] 6-1. Condensation Reaction Step In this step, a C-protected peptide, an N-Fmoc-protected amino acid, and a condensation agent (preferably a condensation agent and an activator) are mixed in a solvent to obtain a C-protected-N-Fmoc-protected peptide with an extended number of amino acid residues. The method and order of adding each component are not particularly limited, and any method commonly used in a condensation step in peptide synthesis can be used.

[0136] The amount of N-Fmoc-protected amino acid used relative to the C-protected peptide is usually 1.01 to 4 equivalents, preferably 1.03 to 3 equivalents, more preferably 1.05 to 2 equivalents, and even more preferably 1.1 to 1.5 equivalents. If the amount is less than this range, unreacted protected peptide is likely to remain, and loss of amino acids is likely to occur.

[0137] As the condensing agent, condensing agents generally used in peptide synthesis can be used without limitation in the present invention, and examples thereof include, but are not limited to, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorphonium chloride (DMT-MM), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU(6-Cl)), Examples of the condensing agent include O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), diisopropylcarbodiimide (DIPCI), dicyclohexylcarbodiimide (DCC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC). Preferred are DMT-MM, HBTU, HATU, or COMU. The amount of the condensing agent used is usually 1 to 4 equivalents, preferably 1 to 2 equivalents, and more preferably 1.05 to 1.3 equivalents relative to the carrier-protected peptide.

[0138] In the condensation step, an activator is preferably added to promote the reaction and suppress side reactions such as racemization. Here, the activator is a reagent that, in the presence of a condensation agent, converts an amino acid into the corresponding activated ester, symmetrical acid anhydride, or the like, thereby facilitating the formation of a peptide bond (amide bond). Examples of activators that are commonly used in peptide synthesis can be used without limitation in the present invention. Examples include HOBt, HOCt, HOAt, HOOBt, HOSu, HOPht, HONb, pentafluorophenol, and ethyl cyanohydroxyiminoacetate (Oxyma). Preferred are HOBt, HOOBt, HOCt, HOAt, HONb, HOSu, and Oxyma. The amount of activator used is typically 1 to 4 equivalents, preferably 1 to 2 equivalents, and more preferably 1.05 to 1.3 equivalents relative to the carrier-protected peptide. The solvent used in the condensation step may be any solvent commonly used in peptide synthesis, including, but not limited to, the solvents described above. The amount of solvent used is such that the concentration of the carrier-protected peptide or the like dissolved therein is generally 0.1 mM to 1 M, preferably 1 mM to 0.5 M.

[0139] The reaction temperature used in the present invention is the same as that generally used in peptide synthesis, and is usually within the range of, for example, −20 to 40° C., preferably 0 to 30° C. The reaction time (time for one cycle) is usually 0.5 to 30 hours.

[0140] 6-2. Scavenging Reaction Step The peptide synthesis method of the present invention can further include a step of adding a water-soluble amine to the reaction system after the amino acid condensation reaction step to scavenge (capture) unreacted amino acid activated esters. In this step, the water-soluble amine bonds with the amino acid activated ester to form a scavenger, thereby inactivating the activated ester. In this specification, the water-soluble amine used in the present invention may also be referred to as an amine scavenger. The water-soluble amine usable as a scavenger in the present invention is preferably a divalent or higher water-soluble amine having at least one primary or secondary amino group, such as 1-methylpiperazine, 4-aminopiperidine, diethylenetriamine, triaminoethylamine, 1-ethylpiperazine, N,N-dimethylethylenediamine, ethylenediamine, and piperazine, preferably 1-methylpiperazine, 4-aminopiperidine, diethylenetriamine, N,N-dimethylethylenediamine, and ethylenediamine, more preferably 1-methylpiperazine, 4-aminopiperidine, N,N-dimethylethylenediamine, and diethylenetriamine, and even more preferably 1-methylpiperazine. Here, the divalent or higher water-soluble amine refers to a water-soluble amine having two or more amino groups.

[0141] The amount of water-soluble amine added in step (ii) is typically 1 to 10 equivalents, preferably 1 to 6 equivalents, and more preferably 1 to 4 equivalents, relative to the theoretically remaining amino acid equivalent. If the amount of amine added is less than this range, scavenging (capturing) of the amino acid activated ester is insufficient, resulting in a double hit reaction between the remaining amino acid activated ester and the amino group regenerated in the subsequent step (iii), reducing purity and yield. On the other hand, if the amount of amine added is greater than this range, the Fmoc deprotection reaction proceeds simultaneously, resulting in a double hit reaction between the remaining amino acid activated ester and the regenerated amino group, reducing purity and yield. By adding this step to the peptide synthesis method of the present invention, the subsequent step of removing the Fmoc group from the N-Fmoc-protected peptide can be performed after scavenging (capturing) the amino acid activated ester in the reaction system with an amine scavenger to form a scavenger. This allows the amino acid activated ester in the reaction solution to be inactivated during the Fmoc deprotection reaction, preventing double hits of the amino acid during deprotection without the need to remove them from the reaction system. Furthermore, the amino acid active ester trapped in the water-soluble amine can be easily removed in the subsequent washing step.

[0142] 6-3. Fmoc Removal Step In the peptide synthesis method of the present invention, the Fmoc group is removed from the C-protected-N-Fmoc-protected peptide. For the removal of the Fmoc group from the N-terminus, a removal method commonly used in peptide synthesis can be used in the present invention with appropriate modifications as necessary. Examples of Fmoc removal reagents that can be used in the present invention include, but are not limited to, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1,5-diazabicyclo[4.3.0]-5-nonene (DBN), 1,4-diazabicyclo[2.2.2]-octane (DABCO), triethylamine, and tributylamine, with DBU being preferred.

[0143] The removal of the Fmoc group is preferably carried out in the presence of a water-soluble amine, and further, it is preferable to additionally add a water-soluble amine to the reaction system in this step. The water-soluble amine that can be used in this step is preferably a divalent or higher water-soluble amine having at least one primary or secondary amino group, such as 1-methylpiperazine, 4-aminopiperidine, diethylenetriamine, triaminoethylamine, 1-ethylpiperazine, N,N-dimethylethylenediamine, ethylenediamine, or piperazine. Preferred are 1-methylpiperazine, 4-aminopiperidine, and diethylenetriamine, and more preferably 1-methylpiperazine. The water-soluble amine used in this step may be the same as or different from the water-soluble amine added in step (ii) (the scavenging reaction step). The equivalent amount of the water-soluble amine added in step (iii) is 5 to 30 equivalents, preferably 5 to 20 equivalents, and more preferably 10 to 20 equivalents, relative to the amount of Fmoc groups present in the system. If the amount of amine added is less than this range, scavenging (capture) of dibenzofulvene (DBF) generated by the Fmoc deprotection reaction will be insufficient, making it difficult to remove the impurity in the subsequent acidic aqueous solution washing step. On the other hand, if the amount of amine added is greater than this range, the amount of acid required for neutralization will increase, and the accompanying neutralization step will cause side reactions (decomposition, racemization), resulting in a decrease in purity and a decrease in yield.

[0144] In this step, the Fmoc deprotection reaction is carried out in the presence of a water-soluble amine. However, if the water-soluble amine present in the system functions as an Fmoc deprotection reagent, it is not necessary to add another Fmoc deprotection reagent to the system. On the other hand, to efficiently carry out the Fmoc deprotection reaction, another Fmoc deprotection reagent may be added to the system. Examples of water-soluble amines that function as Fmoc deprotection reagents include the water-soluble amines exemplified above. Preferably, in this step, the Fmoc deprotection reagent is added together with the water-soluble amine. When the Fmoc deprotection reagent is added to the reaction system together with the water-soluble amine in this step, the water-soluble amine and the Fmoc deprotection reagent may be added simultaneously, or the water-soluble amine may be added to the system followed by the Fmoc deprotection reagent. Here, "simultaneous" includes additions that are considered to be simultaneous in the context of reactions in this technical field. When the water-soluble amine is added to the system followed by the Fmoc deprotection reagent, the time interval between additions can be adjusted appropriately, taking into account the operation and other factors. During the Fmoc removal reaction, DBF is generated, but the water-soluble amine added in this step can scavenge (capture) this impurity. The DBF captured by the water-soluble amine can be easily removed in the subsequent acidic aqueous solution washing step.

[0145] 6-4. Acidic Aqueous Solution Washing Step The neutralization step (iv) converts excess base and scavengers present in the system into salts, thereby improving their water solubility. The acid used for neutralization is not particularly limited as long as it can neutralize the base in the reaction solution, and examples include aqueous solutions of hydrogen chloride, phosphoric acid, acetic acid, sulfuric acid, etc. For example, when hydrochloric acid is used, although this is not limited thereto, 1 N to 12 N, preferably 2 N to 12 N, and more preferably 5 N to 12 N hydrochloric acid is added. Neutralization here refers to the reaction solution being brought to a neutral pH, and may be a pH of 7.0 or less.

[0146] In step (iv), an acidic aqueous solution is further added to the neutralized reaction solution neutralized with acid, followed by washing. The solution is then separated, the aqueous layer is discarded, and the organic layer is recovered. This allows impurities soluble in the acidic aqueous solution to be removed. The acidic aqueous solution used is not particularly limited, but examples include hydrochloric acid, dilute sulfuric acid, phosphoric acid, and acetic acid, with hydrochloric acid being preferred. The pH of the acidic aqueous solution is 1 to 5, preferably 1 to 4, and more preferably 1 to 3. The amount of the acidic aqueous solution used for washing is not particularly limited as long as it provides a cleaning effect, but it can be used in an amount of 0.1 to 3 times, preferably 0.5 to 2 times, and more preferably 0.8 to 1.5 times the amount of the reaction solution. The washing, separation, and aqueous layer discarding steps can be performed once or multiple times without any restrictions. The number of times can be appropriately selected depending on the type of compound and amount of impurities in the reaction system, and the purpose.

[0147] In the present invention, impurities can be removed by washing with an acidic aqueous solution in step (iv). The separation of the acidic aqueous solution can remove impurities such as HN-AAx-amine (scavenger) conjugates (where AA represents an amino acid and x represents its number), decomposition products of condensing agents, pH-adjusting bases, DBF-amine (scavenger) conjugates, amine (scavenger), and Fmoc deprotection reagents, thereby obtaining a supported peptide dissolved in a reaction system with reduced or removed impurities. Furthermore, separation using an aqueous solution is simple and contributes to shortening the process time. Furthermore, solid-liquid separation is not required, reducing the use of poor solvents for insolubilizing the supported peptide. In continuous peptide synthesis using the method of the present invention, in the final cycle after the Fmoc deprotection step, the supported peptide may be neutralized with acid, insolubilized, and then recovered using solid-liquid separation. However, washing with an acidic aqueous solution is preferred from the perspective of more complete removal of impurities.

[0148] One embodiment of the peptide synthesis method of the present invention includes a washing step with an acidic aqueous solution to reduce or remove impurities generated in the condensation step and the Fmoc removal step. However, other washing steps may be added as long as they do not prevent the C-carrier protected peptide from being recovered in a dissolved state in an organic solvent. Examples of such washing steps include washing with a weakly basic aqueous solution or washing with saline. Alternatively, dehydration can be performed by adding a dehydrating agent such as anhydrous sodium sulfate instead of washing with saline. Examples of washing with a weakly basic aqueous solution include washing with an aqueous sodium bicarbonate solution, an aqueous sodium carbonate solution, or an aqueous potassium carbonate solution at a pH of 8 to 12 (preferably 8 to 10).

[0149] 7. Crystallization and Separation Step of C-Supported Peptide The C-supported peptide synthesized by the peptide synthesis method of the present invention can be insolubilized (e.g., crystallized or oiled) and separated after neutralization with an acid in step (iv), preferably after the liquid separation procedure using an acidic aqueous solution in step (iv). Alternatively, the C-supported peptide synthesized by repeating steps (i) to (iv) as many times as necessary using the method of the present invention can be insolubilized and separated after any step after step (i) without performing step (iii) if the N-terminus is not deprotected after step (i). Insolubilization can be achieved by concentrating the solvent in which the C-supported peptide is dissolved to solidify it, or by changing the solvent composition to cause insolubilization (crystallization or oiling). Insolubilization by changing the solvent composition can be achieved by appropriately referring to methods known in the field of peptide synthesis using a carrier that has the property of reversibly switching between a dissolved state and an insolubilized state.

[0150] 8. Carrier Deprotection Step Carrier deprotection is carried out by cleaving the carrier bound to the peptide directly or via a linker in the C-carrier-protected peptide synthesized by the method of the present invention. When the carrier is bound directly to the peptide, carrier deprotection can be carried out by removing (deprotecting) the carrier bound to the carboxyl group of the synthesized peptide. There are no particular limitations on the method for removing the carrier, and any known deprotection method may be used, but acid treatment is preferred. For example, a deprotection method using TFA can be used. More specifically, deprotection is preferably carried out using 50 to 100% trifluoroacetic acid at a concentration varying depending on the type of carrier used.

[0151] When the support is bound to the peptide via a linker, the support can be deprotected either by (i) cleaving the bond between the linker and the peptide, or by (ii) cleaving the bond between the linker and the support. In the latter case, the peptide is cleaved from the support while still retaining the linker, thereby obtaining a peptide whose terminus or side chain is modified by the linker. In the case of (i), support deprotection can be carried out by a deprotection method using TFA, for example, when the linker and the peptide are bound via an ester bond or an amide bond. In the case of (ii), the support can be deprotected using the same deprotection method as in the case of direct binding described above.

[0152] 9. Synthesis of C-Terminally Modified Peptides C-terminal amidation is a modification frequently observed in biologically active peptides, such as calcitonin, castrin, secretin, and hormone-releasing factor. By using the peptide synthesis method of the present invention and a carrier having a reactive group containing an amino group, peptides with an amidated C-terminus can be efficiently synthesized. For example, in the method of the present invention, a C-carrier-protected amino acid (or peptide) in which a compound (carrier) of the present invention having an amino group that bonds to the carboxyl group as a reactive group is bound to the carboxyl terminus of any amino acid or peptide can be used to obtain peptides with an amidated C-terminus, e.g., modified with an amino group or aminoalkyl group. Examples of the amide carrier include compounds of the present invention having an amino group or alkylamino group as a reactive group.

[0153] By using such a compound (carrier) of the present invention, a peptide whose C-terminus is modified with an amino group or an alkylamino group can be obtained. For example, but not limited to, a peptide in which the C-terminal proline is aminoethylated can be obtained. In one embodiment of the present invention, the method of the present invention using an amide carrier is useful for synthesizing a peptide whose C-terminus is amidated.

[0154] The peptide obtained by the peptide synthesis method of the present invention can be isolated and purified according to a method commonly used in peptide synthesis, for example, by subjecting the reaction mixture to extraction and washing, crystallization, chromatography, etc., to isolate and purify the target peptide.

[0155] Although there are no particular limitations on the peptides produced by the peptide synthesis method of the present invention, it is preferable that the number of amino acid residues of the peptide is, for example, not more than several tens. Like existing or unknown synthetic peptides and natural peptides, the peptides obtained by the peptide synthesis method of the present invention can be used in various fields, including, but not limited to, pharmaceuticals, foods, cosmetics, electronic materials, etc.

[0156] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples. A carrier (tag) of the present invention for peptide synthesis was synthesized as in the following examples.

[0157] Synthesis of (3,4,5-tris((8-(nonyloxy)octyl)oxy)phenyl)methanol (Compound 3) and 3,4,5-tris((8-(nonyloxy)octyl)oxy)benzoic acid (Compound 4)

[0158]

[0159] (1) To a suspension of 1.60 g (40 mmol) of sodium hydride in THF (32 mL), 3.48 mL (20 mmol) of 1-nonanol and 7.4 mL (40 mmol) of 1,8-dibromooctane were added, and the mixture was heated for 70 minutes under an argon atmosphere. o The mixture was stirred at RT for 24 hours. The reaction solution was returned to room temperature, and 30 mL of saturated brine was added. The organic layer was extracted three times with 30 mL of ethyl acetate, and the extracted organic layer was washed once with 30 mL of saturated brine. The washed organic layer was then dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (100% hexane to 60:1 hexane:ethyl acetate) to obtain 4.9 g of compound 1 (76% yield).

[0160] (2) 0.55 g (3.0 mmol) of methyl gallate was dissolved in 30 mL of DMF, and 4.9 g (15.2 mmol) of compound 1 and 1.2 g (9.0 mmol) of potassium carbonate were added to the solution. o The mixture was stirred at RT for 21 hours. After filtering the stirred solution to remove potassium carbonate, 18% brine was added. The organic layer was extracted three times with 30 mL of ethyl acetate, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 20:1) to obtain 2.2 g of compound 2 (77% yield).

[0161] (3) 2.3 g (2.4 mmol) of compound 2 was dissolved in 24 mL of THF, and 127.5 mg (3.4 mmol) of LiAlH4 was added in an ice bath. The reaction temperature was returned to room temperature and stirred under an Ar atmosphere for 4 hours. 25 mL of water and 11 mL of 1 N hydrochloric acid were then added. The organic layer was extracted three times with 30 mL of hexane. This organic layer was dried over magnesium sulfate, and the solvent was evaporated. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 5:1) to obtain 2.2 g of compound 3 (96% yield). 1 H NMR (CDCl3, 400 MHz) δ 6.56 (2H, s), 4.59 (2H, d, J = 6.0 Hz), 4.02-3.86 (6H, m), 3.39 (12H, t, J = 6.9 Hz), 1.84-1.66 (7H, m), 1.61-1.51 (12H, m), 1.51-1.41 (6H, m), 1.41-1.17 (54H, m), 0.93-0.79 (9H, m); 13 C NMR (CDCl3, 101 MHz) δ 153.2, 137.6, 136.1, 105.4, 73.3, 71.0, 70.9, 69.0, 65.6, 31.9, 30.3, 29.8, 29.6, 29.5, 29.4, 29.3, 29.3, 29.3, 26.2, 26.1, 26.0, 26.0, 22.7, 14.1

[0162] (4) 2.2 g of compound 2 was dissolved in 23 mL of 2-propanol, and 2.3 mL of 1.0 M aqueous KOH was added to the solution. o The mixture was stirred at RT for 2 hours. 5.0 mL of 6 N hydrochloric acid was added to the solution, and the organic layer was evaporated under reduced pressure. 30 mL of water and 20 mL of THF were added. The organic layer was then extracted three times with 30 mL of ethyl acetate, and this organic layer was dried over magnesium sulfate. The solvent was evaporated under reduced pressure to give 2.2 g (yield quant.) of compound 4. 1H NMR (CDCl3, 600 MHz) δ7.31 (2H, s), 4.07 (6H, m), 3.43-3.34 (12H, m), 1.86 (4H, m), 1.77-1.69 (2H, m), 1.62-1.52 (12H, m), 1.52-1.42 (6H, m), 1.41-1.20 (54H, m), 0.87 (9H, t, J = 6.9 Hz); 13 C NMR (CDCl3, 151 MHz) δ 171.1, 152.8, 143.0, 123.7, 108.5, 73.5, 71.0, 70.9, 70.9, 69.1, 31.9, 30.3, 29.8, 29.6, 29.5, 29.5, 29.4, 29.3, 29.3, 29.2, 26.2, 26.2, 26.0, 22.7, 14.1.

[0163] Synthesis of (3,4,5-tris((12-(pentyloxy)dodecyl)oxy)phenyl)methanol (compound 7) and 3,4,5-tris((12-(pentyloxy)dodecyl)oxy)benzoic acid (compound 8)

[0164]

[0165] (1) To a suspension of 800 mg (20 mmol) of sodium hydride in THF (16 mL), 1.08 mL (10 mmol) of 1-pentanol and 6.56 g (20 mmol) of 1,12-dibromododecane were added, and the mixture was heated for 70 minutes under an argon atmosphere. o The mixture was stirred at RT for 24 hours. The reaction mixture was returned to room temperature, and 40 mL of water was added. The organic layer was extracted three times with 30 mL of ethyl acetate. This organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (hexane 100% to hexane:ethyl acetate = 40:1) to obtain 2.52 g of compound 5 (yield 38%).

[0166] (2) 184 mg (1.0 mmol) of methyl gallate was dissolved in 30 mL of DMF, and 1.4 g (4.2 mmol) of compound 5 and 1.1 g (8.0 mmol) of potassium carbonate were added to the solution. The mixture was stirred for 90 minutes under an argon atmosphere. o The mixture was stirred overnight at 100°C. After stirring, potassium carbonate was removed by filtration, and 30 mL of water was added. The organic layer was extracted three times with 20 mL of ethyl acetate, and this organic layer was washed once with 30 mL of saturated brine. The washed organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (hexane:ethyl acetate=20:1) to obtain 649 mg of compound 6 (71% yield).

[0167] (3) 235 mg (0.248 mmol) of compound 6 was dissolved in 5 mL of THF, and 14.5 mg (0.383 mmol) of LiAlH4 was added in an ice bath. After stirring for 2 hours in an ice bath, 20 μL of water and 20 μL of 15% aqueous sodium hydroxide solution were added, followed by stirring and an additional 45 μL of water. The solution was dried over sodium sulfate, filtered through Celite, and the solvent was evaporated. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1) to give 195 mg of compound 7 (86% yield). 1 H NMR (CDCl3, 600 MHz) δ 6.56 (2H, s), 4.59 (2H, d, J = 5.5 Hz), 3.97 (4H, t, J = 6.9 Hz), 3.93 (2H, t, J = 6.9 Hz), 3.39 (12H, t, J = 6.9 Hz), 1.86-1.76 (4H, m), 1.76-1.68 (3H, m), 1.60-1.51 (12H, m), 1.50-1.40 (6H, m), 1.39-1.22 (54H, m), 0.94-0.85 (9H, m); 13C NMR (CDCl3, 151 MHz) δ 153.3, 137.6, 136.0, 105.4, 73.4, 71.0, 70.9, 69.1, 65.6, 30.3, 29.8, 29.7, 29.7, 29.7, 29.6, 29.5, 29.5, 29.5, 29.4, 28.4, 26.2, 26.1, 26.1, 22.5, 14.0.

[0168] (4) 182 mg (0.193 mmol) of compound 6 was dissolved in 3 mL of THF, and 2.0 mL of 1.0 M LiOH aq. was added. o The mixture was stirred at C for 6 days. 20 mL of 6 N hydrochloric acid was added to the solution, and the organic layer was extracted three times with 10 mL of ethyl acetate. This organic layer was dried over sodium sulfate. The solvent was evaporated under reduced pressure to give 171 mg of compound 8 (yield 95%). 1 H NMR (CDCl3, 600 MHz) δ 7.32 (2H, s), 4.09-3.97 (6H, m), 3.45-3.35 (12H, m), 1.86-1.78 (4H, m), 1.78-1.69 (2H, m), 1.63-1.52 (12H, m), 1.51-1.41 (6H, m), 1.40-1.22 (54H, m), 0.95-0.85 (9H, m); 13 C NMR (CDCl3, 151 MHz) δ 171.2, 152.7, 142.8, 124.1, 108.4, 73.4, 70.9, 70.9, 69.1, 30.3, 29.7, 29.7, 29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 29.2, 28.3, 26.1, 26.0, 26.0, 22.5, 14.0171.3, 152.9, 143.0, 124.2, 108.6, 73.6, 71.1, 71.0, 69.2, 30.4, 29.9, 29.8, 29.8, 29.7, 29.7, 29.6, 29.5, 29.5, 29.4, 28.5, 26.3, 26.2, 26.2, 22.7, 14.1

[0169] Synthesis of (3,4,5-tris(4-(tridecyloxy)butoxy)phenyl)methanol (Compound 11) and 3,4,5-tris(4-(tridecyloxy)butoxy)benzoic acid (Compound 12)

[0170]

[0171] (1) To a suspension of 1.60 g (40 mmol) of sodium hydride in THF (32 mL), 4.01 g (20 mmol) of 1-tridodecanol and 4.72 mL (40 mmol) of 1,4-dibromobutane were added, and the mixture was heated for 70 minutes under an argon atmosphere. o The mixture was stirred overnight at C. The reaction solution was returned to room temperature, and 50 mL of water was added. The organic layer was extracted five times with 50 mL of ethyl acetate, and this organic layer was washed once with 50 mL of saturated brine. The organic layer was then dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (100% hexane to 60:1 hexane:ethyl acetate) to obtain 3.8 g of compound 9 (yield 56%).

[0172] (2) 177.1 mg (0.93 mmol) of methyl gallate was dissolved in 3.1 mL of DMF, and 1.4 g (4.18 mmol) of compound 9 and 385.3 mg (2.79 mmol) of potassium carbonate were added to the solution. o The mixture was stirred overnight at C. After stirring, potassium carbonate was removed from the solution by filtration, and purified water was added. The organic layer was extracted eight times with 50 mL of ethyl acetate, and the organic layer was dried over sodium sulfate, after which the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (hexane 100% to hexane:ethyl acetate = 5:1) to obtain 660 mg of compound 10 (yield 75%).

[0173] (3) 142.1 mg (0.15 mmol) of compound 10 was dissolved in 1.5 mL of THF, and 17.0 mg (0.45 mmol) of LiAlH4 was added in an ice bath. The reaction mixture was allowed to return to room temperature while stirring for 4 hours, and then 4.5 mL of water and 1.5 mL of 1 N hydrochloric acid were added. The organic layer was extracted three times with 30 mL of hexane. This organic layer was dried over sodium sulfate, and the solvent was then evaporated. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 3:1) to obtain 125 mg of compound 11 (91% yield). 1 H NMR (CDCl3, 600 MHz) δ 6.55 (2H, s), 4.57 (2H, s), 3.99 (4H, t, J = 6.9 Hz), 3.96-3.92 (2H, m), 3.49-3.44 (6H, m), 3.42-3.36 (6H, m), 2.03 (1H, s), 1.90-1.83 (4H, m), 1.82-1.70 (8H, m), 1.59-1.52 (6H, m), 1.37-1.20 (60H, m), 0.88 (9H, t, J = 6.9 Hz); 13 C NMR (CDCl3, 151 MHz) δ 153.1, 137.3, 136.2, 105.2, 73.0, 71.0, 70.9, 70.6, 70.3, 68.7, 65.4, 31.9, 29.8, 29.7, 29.7, 29.6, 29.5, 29.5, 29.3, 27.0, 26.3, 26.2, 26.2, 22.6, 14.1

[0174] (4) 142.1 mg of compound 10 was dissolved in 1.5 mL of 2-propanol, and 150 μL of 1.0 M KOH aqueous solution was added to the solution for 60 minutes. o The mixture was stirred at C for 23 hours. 1.6 mL of 1 N hydrochloric acid was added to this solution, and the organic layer was evaporated under reduced pressure, followed by the addition of 30 mL of water and 20 mL of THF. The organic layer was then extracted three times with 30 mL of ethyl acetate, and this organic layer was dried over sodium sulfate. The solvent was evaporated under reduced pressure to give 80 mg of compound 12 (yield 57%). 1H NMR (CDCl3, 600 MHz) δ 7.31 (2H, m), 4.08-4.01 (6H, m), 3.50-3.44 (6H, m), 3.43-3.37 (6H, m), 1.93-1.86 (4H, m), 1.84-1.71 (8H, m), 1.61-1.51 (6H, m), 1.36-1.20 (60H, m), 0.88 (9H, t, J = 6.9 Hz); 13 C NMR (CDCl3, 151 MHz) δ 171.3, 152.7, 142.9, 123.9, 108.5, 73.2, 71.0, 71.0, 70.5, 70.3, 68.9, 31.9, 29.8, 29.7, 29.7, 29.6, 29.5, 29.5, 29.3, 27.0, 26.3, 26.3, 26.2, 26.2, 22.7, 14.1

[0175] Synthesis of (3,4,5-tris((10-(undecyloxy)decyl)oxy)phenyl)methanol (Compound 15) and 3,4,5-tris((10-(undecyloxy)decyl)oxy)benzoic acid (Compound 16)

[0176]

[0177] (1) To a suspension of 1.60 g (40 mL) of sodium hydride in THF (32 mL), 4.1 mL (20 mmol) of 1-undecanol and 9.0 mL (40 mmol) of 1,10-dibromodecane were added, and the mixture was heated for 70 minutes under an argon atmosphere. o The mixture was stirred at C for 24 hours. The reaction solution was returned to room temperature, and 10 mL of HO was added. The organic layer was extracted three times with 30 mL of ethyl acetate, and this organic layer was washed once with 20 mL of saturated brine. The organic layer was then dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (hexane:ethyl acetate = 80:1) to obtain 4.4 g of compound 13 (yield 55%).

[0178] (2) 0.46 g (2.5 mmol) of methyl gallate was dissolved in 12.5 mL of DMF, and 4.4 g (11.3 mmol) of compound 13 and 1.5 g (11.3 mmol) of potassium carbonate were added to the solution. o The mixture was stirred at RT for 18 hours. 20 mL of THF was added to the stirred solution, and potassium carbonate was removed by filtration. 18% brine was then added. The organic layer was extracted three times with 30 mL of THF, and this organic layer was washed with 18% brine. The organic layer was then dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (hexane:ethyl acetate=20:1) to obtain 0.82 g of compound 14 (yield 29%).

[0179] (3) 0.22 g (0.2 mmol) of compound 14 was dissolved in 4 mL of THF, and 15.2 mg (0.4 mmol) of LiAlH4 was added in an ice bath. The reaction mixture was stirred for 1 hour while the temperature was returned to room temperature, and then 10 mL of HO was added in an ice bath. After removing the precipitate from the solution by filtration, 10 mL of saturated brine was added, and the organic layer was extracted three times with 20 mL of ethyl acetate. The organic layer was dried over sodium sulfate, and the solvent was evaporated to give 0.21 g of compound 15 (yield 99%). 1 H NMR (CDCl3, 600 MHz) δ 6.56 (2H, s), 4.59 (2H, d, J = 4.8 Hz), 3.97 (4H, t, J = 6.9 Hz), 3.93 (2H, t, J = 6.9 Hz), 3.39 (12H, t, J = 6.9 Hz), 1.83-1.65 (7H, m), 1.60-1.51 (12H, m), 1.50-1.40 (6H, m), 1.39-1.20 (78H, m), 0.88 (9H, t, J = 6.9 Hz); 13C NMR (CDCl3, 151 Hz) δ 153.3, 137.6, 136.0, 105.4, 73.4, 71.0, 69.1, 65.7, 31.9, 30.3, 29.8, 29.6, 29.6, 29.5, 29.4, 29.3, 26.2, 26.2, 26.1, 26.0, 22.7, 14.1.

[0180] (4) 0.22 mg (0.2 mmol) of compound 14 was dissolved in 6 mL of THF, and 2 mL of a 1 M LiOH aqueous solution was added to the solution, and the mixture was stirred for 70 minutes. o The mixture was stirred at RT for 39 hours at RT. 0.5 mL of 6 N hydrochloric acid was added to the stirred solution, and the organic layer was extracted three times with 20 mL of ethyl acetate. This organic layer was washed with 20 mL of saturated brine. The washed organic layer was dried over sodium sulfate, and the solvent was evaporated under reduced pressure to give 177.1 mg of compound 16 (yield 80%). 1 H NMR (CDCl3, 600 MHz) δ 7.31 (2H, s), 4.06-3.98 (6H, m), 3.43-3.34 (12H, m), 1.85-1.77 (4H, m), 1.77-1.70 (2H, m), 1.61-1.52 (12H, m), 1.51-1.42 (6H, m), 1.41-1.18 (78H, m), 0.88 (9H, t, J = 6.9 Hz); 13 C NMR (CDCl3, 151 Hz) δ 170.7, 152.8, 143.1, 123.7, 108.6, 73.5, 71.0, 71.0, 69.2, 31.9, 30.3, 29.8, 29.7, 29.6, 29.6, 29.5, 29.3, 29.2, 26.2, 26.2, 26.0, 26.0, 22.7, 14.1.

[0181] Synthesis of (3,4,5-tris(4-((7-(pentyloxy)heptyl)oxy)butoxy)phenyl)methanol (compound 20) and 3,4,5-tris(4-((7-(pentyloxy)heptyl)oxy)butoxy)benzoic acid (compound 21)

[0182]

[0183] (1) To a suspension of 1.60 g (40 mmol) of sodium hydride in THF (72 mL), 5.5 mL (40 mmol) of 1,7-heptanediol and 2.6 mL (20 mmol) of 1-iodopentane were added, and the mixture was heated for 70 minutes under an argon atmosphere. o The mixture was stirred at C for 25 hours. After stirring, the reaction solution was returned to room temperature and 30 mL of HO was added. The organic layer was extracted three times with 30 mL of ethyl acetate, and this organic layer was washed once with 30 mL of saturated brine. The washed organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (hexane:ethyl acetate = 3:1) to obtain 1.8 g of compound 17 (44% yield).

[0184] (2) To a suspension of 704 mg (17.6 mmol) of sodium hydride in THF (14 mL), 1.8 g (8.8 mmol) of compound 17 and 2.1 mL (17.6 mmol) of 1,4-dibromobutane were added, and the mixture was stirred for 70 minutes under an argon atmosphere. o The mixture was stirred at RT for 19 hours. After stirring, the reaction mixture was returned to room temperature and 20 mL of HO was added. The organic layer was extracted three times with 30 mL of ethyl acetate, and this organic layer was washed once with 20 mL of saturated brine. The organic layer was then dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (hexane:ethyl acetate = 20:1 to 10:1) to obtain 1.2 g of compound 18 (39% yield).

[0185] (3) Methyl gallate 138.1 (0.75 mmol) was dissolved in 7.5 mL of DMF, and 1.2 g (3.4 mmol) of compound 18 and 317.6 mg (2.3 mmol) of potassium carbonate were added to the solution. oThe mixture was stirred at RT for 19 hours at RT. After the stirring, potassium carbonate was removed from the solution by filtration, and 20 mL of saturated brine was added. The organic layer was extracted three times with 30 mL of ethyl acetate, and this organic layer was washed with 20 mL of saturated brine. After drying over magnesium sulfate, the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (hexane:ethyl acetate = 7:1 to 5:1) to obtain 602.0 mg of compound 19 (yield 84%).

[0186] (4) 95.3 mg (0.1 mmol) of compound 19 was dissolved in 1 mL of THF, and 7.6 mg (0.2 mmol) of LiAlH4 was added in an ice bath. The solution was stirred for 1 hour while the reaction temperature was returned to room temperature, and then 10 mL of H2O was added and the precipitate was removed by filtration. The organic layer was extracted twice with 20 mL of ethyl acetate. This organic layer was dried over sodium sulfate, and the solvent was evaporated to give 103.4 mg (yield quant.) of compound 20. 1 H NMR (CDCl3, 600 MHz) δ 6.56 (2H, s), 4.59 (2H, s), 4.00 (4H, t, J = 6.9 Hz), 3.95 (2H, t, J = 6.2 Hz), 3.49-3.43 (6H, m), 3.42-3.34 (18H, m), 1.91 (4H, m), 1.82-1.66 (9H, m), 1.61-1.49 (18H, m), 1.39-1.22 (30H, m), 0.94-0.85 (9H, m); 13 C NMR (CDCl3, 151 Hz) δ 153.1, 137.4, 136.2, 105.3, 73.0, 71.0, 70.9, 70.6, 70.4, 68.8, 65.5, 29.8, 29.7, 29.4, 29.4, 29.4, 28.4, 27.0, 26.4, 26.3, 26.3, 26.2, 22.5, 14.0.

[0187] (5) 95.3 mg (0.1 mmol) of compound 19 was dissolved in 3.3 mL of THF-MeOH-H2O (1:2:1.5), and 44 mg of lithium hydroxide was added to the solution. oThe mixture was stirred at C for 48 hours. 1.5 mL of 6 N hydrochloric acid was added to the stirred solution, and the organic layer was extracted three times with 15 mL of cyclohexane. This organic layer was dried over sodium sulfate. The solvent was evaporated under reduced pressure from the dried solution, yielding 97.1 mg (yield quant.) of compound 21. 1 H NMR (CDCl3, 600 MHz) δ 7.31 (2H, s), 4.10-4.01 (6H, m), 3.50-3.44 (6H, m), 3.43-3.34 (18H, m), 1.94-1.85 (4H, m), 1.84-1.71 (8H, m), 1.62-1.50 (18H, m), 1.40-1.27 (30H, m), 0.94-0.84 (9H, m); 13 C NMR (CDCl3, 101 Hz) δ 170.4, 152.6, 142.5, 124.4, 108.4, 73.1, 71.0, 70.9, 70.9, 70.5, 70.4, 68.9, 29.7, 29.6, 29.4, 29.3, 28.3, 27.0, 26.2, 26.1, 26.1, 22.5, 14.0.

[0188] Synthesis of (3,4,5-tris((2,5,8,11-tetraoxatridecan-13-yl)oxy)phenyl)methanol (Compound 23) and 3,4,5-tris((2,5,8,11-tetraoxatridecan-13-yl)oxy)benzoic acid (Compound 24)

[0189]

[0190] (1) 766.7 mg (4.2 mmol) of methyl gallate was dissolved in 21 mL of DMF, and 3.88 mL (18.5 mmol) of triethylene glycol 2-bromoethyl methyl ether and 5.23 g (37.9 mmol) of potassium carbonate were added. The mixture was heated at 90°C under an Ar atmosphere. oThe mixture was stirred at C for 26 hours. 100 mL of water was added to the stirred solution, and the organic layer was extracted five times with 50 mL of ethyl acetate and washed with 250 mL of saturated brine. The organic layer was dried over magnesium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by column chromatography (hexane:ethyl acetate = 1:1 → ethyl acetate:methanol = 3:1) to obtain a crude product containing compound 22. For reactions (2) and (3), 100 mL of water was added to the crude product, and the product was extracted five times with 40 mL of ethyl acetate.

[0191] (2) 435.3 mg (0.58 mmol) of compound 22 was dissolved in 12 mL of THF, and 38.4 mg (1.0 mmol) of LiAlH4 was added in an ice bath. The mixture was stirred under an Ar atmosphere for 2 hours. 39 μL of water and 39 μL of saturated aqueous NaOH were added to the stirred solution, followed by 80 μL of water and thorough stirring. The stirred solution was dried over magnesium sulfate, filtered to remove solids, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 1:3 → ethyl acetate:methanol = 1:1) to obtain 292.0 mg of compound 23 (70% yield). 1 H NMR (CDCl3, 600 MHz) δ 6.63 (2H, s), 4.57 (2H, s), 4.17 (4H, t, J = 4.8 Hz), 4.13 (2H, t, J = 4.8 Hz), 3.84 (4H, t, J = 4.8 Hz), 3.78 (2H, t, J = 4.8 Hz), 3.73-3.61 (30H, m), 3.57-3.51 (6H, m), 3,39-3.34 (9H, m), 1.83 (1H, brs); 13 C NMR (CDCl3, 151 MHz) δ 152.7, 137.8, 136.7, 106.7, 72.2, 71.9, 70.8, 70.7, 70.6, 70.6, 70.6, 70.5, 70.5, 69.8, 68.9, 65.2, 59.0.

[0192] (3) 215.5 mg (0.29 mmol) of compound 23 was dissolved in 3.2 mL of THF, and 3.2 mL of 1.0 M aqueous LiOH was added to the solution. o The mixture was stirred at C for 47 hours. 10 mL of 1 N hydrochloric acid was added to the stirred solution, and the organic layer was extracted four times with 20 mL of ethyl acetate and washed twice with 100 mL of saturated brine. The organic layer was dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:methanol = 1:1) to obtain 105.0 mg of compound 24 (yield 50%). 1 H NMR (CDCl3, 600 MHz) δ 7.39 (2H, s), 4.26-4.18 (6H, m), 3.84 (4H, t, J = 4.8 Hz), 3.80 (2H, t, J = 4.8 Hz), 3.74-3.62 (30H, m), 3.59-3.53 (6H, m), 3.40 (6H, s), 3.38 (3H, m); 13 C NMR (CDCl3, 151 MHz) δ 169.5, 152.2, 142.9, 124.8, 109.8, 72.4, 71.9, 70.8, 70.7, 70.6, 70.6, 70.5, 70.5, 70.5, 70.4, 69.7, 68.9, 59.0, 59.0.

[0193] Synthesis of (2,4-bis((8-(nonyloxy)octyl)oxy)phenyl)methanol (Compound 26) and 2,4-bis((8-(nonyloxy)octyl)oxy)benzoic acid (Compound 27)

[0194]

[0195] (1) 138 mg (1.0 mmol) of 2,4-dihydroxybenzaldehyde was dissolved in 7 mL of DMF, and 900 mg (2.6 mmol) of compound 1 and 760 mg (5.5 mmol) of potassium carbonate were added. The mixture was stirred for 90 minutes under an argon atmosphere. oThe mixture was stirred overnight at 37°C. After the stirring, potassium carbonate was removed from the solution by filtration, and 30 mL of water was added. The organic layer was extracted three times with 20 mL of ethyl acetate. This organic layer was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (hexane:ethyl acetate = 10:1) to obtain 338 mg of compound 25 (yield 67%).

[0196] (2) 98 mg (0.152 mmol) of compound 25 was dissolved in a mixture of 10 mL of THF and 1 mL of IPA, and 11.5 mg (0.304 mmol) of NaBH4 was added. After stirring at room temperature for 2 hours, 20 mL of saturated brine was added. The organic layer was extracted three times with 10 mL of ethyl acetate. This organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 6:1) to obtain 90 mg of compound 26 (91% yield). 1 H NMR (CDCl3, 600 MHz) δ 7.13 (1H, d, J = 8.3 Hz), 6.45 (1H, d, J = 2.1 Hz), 6.42 (1H, dd, J = 8.3, 2.1 Hz), 4.61 (2H, d, J = 6.9 Hz), 3.98 (2H, t, J = 6.9 Hz), 3.93 (2H, t, J = 6.9 Hz), 3.42-3.35 (8H, m), 2.23 (1H, t, J = 6.9 Hz), 1.83-1.73 (4H, m), 1.61-1.52 (8H, m), 1.51-1.18 (40H, m), 0.88 (6H, t, J = 6.9 Hz); 13 C NMR (CDCl3, 151 MHz) δ 160.3, 158.3, 129.7, 121.9, 104.6, 99.9, 71.2, 71.1, 68.3, 68.1, 62.2, 32.0, 29.9, 29.9, 29.7, 29.7, 29.6, 29.5, 29.5, 29.4, 29.4, 26.4, 26.3, 26.3, 26.2, 26.2, 22.8, 14.3.

[0197] (3) 338 mg (0.522 mmol) of compound 25 was dissolved in a mixture of 6 mL of THF, 6 mL of t-BuOH, and 4 mL of water. 626 mg (5.22 mmol) of NaH2PO4, 832 μL (7.8 mmol) of 2-methyl-2-butene, and 295 mg (2.61 mmol) of NaClO2 were added and stirred at room temperature for 20 hours. 20 mL of saturated brine was added to the stirred solution, and the organic layer was extracted three times with 50 mL of ethyl acetate. This organic layer was washed with saturated brine and then dried over sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (hexane:ethyl acetate = 5:1) to obtain 13 mg of compound 27 (4% yield). 1 H NMR (CDCl3, 600 MHz) δ 10.62 (1H, brs), 8.11 (1H, d, J = 8.9 Hz), 6.61 (1H, dd, J = 8.9 Hz, 2.1 Hz), 6.50 (1H, d, J = 2.1 Hz), 4.19 (2H, t, J = 6.9 Hz), 4.01 (2H, t, J = 6.9 Hz), 3.43-3.36 (8H, m), 1.95-1.86 (2H, m), 1.83-1.75 (2H , m), 1.61-1.53 ​​(8H, m), 1.52-1.42 (4H, m), 1.42-1.21 (36H, m), 0.88 (6H, t, J = 6.9 Hz); 13 C NMR (CDCl3, 151 MHz) δ 165.3, 164.6, 159.0, 135.4, 110.2, 107.0, 99.8, 71.0, 70.9, 70.9, 70.8, 70.1, 68.5, 63.0, 32.8, 31.9, 29.7, 29.7, 29.6, 29.5, 29.4, 29.4, 29.3, 29.1, 29.0, 28.8, 26.2, 26.1, 26.1, 25.9, 25.8, 25.7, 22.7, 14.1.

[0198] Synthesis of (2,4-bis((10-(undecyloxy)decyl)oxy)phenyl)methanol (Compound 29) and 2,4-bis((10-(undecyloxy)decyl)oxy)benzoic acid (Compound 30)

[0199]

[0200] (1) 414.4 mg (3.0 mmol) of 2,4-dihydroxybenzaldehyde was dissolved in 15 mL of DMF, and 3.6 g (9.1 mmol) of compound 13 and 1.2 g (9.0 mmol) of potassium carbonate were added. o The mixture was stirred at RT for 18 hours at RT. After the stirred solution was filtered to remove potassium carbonate, 40 mL of saturated brine was added. The organic layer was extracted three times with 40 mL of THF, and this organic layer was washed with 40 mL of saturated brine. The mixture was then dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (hexane:ethyl acetate=20:1) to obtain 2.2 g of compound 28 (yield 96%).

[0201] (2) 759.3 mg (1.0 mmol) of compound 28 was dissolved in a mixture of 20 mL of THF and 2 mL of IPA, and 75.6 mg (2.0 mmol) of NaBH4 was added and stirred at room temperature for 3 hours. After stirring, 17% brine was added to the reaction solution, and the organic layer was extracted three times with 30 mL of THF. This organic layer was dried over sodium sulfate. The solvent was then removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane:ethyl acetate = 7:1) to obtain 738.6 mg (97% yield) of compound 29. 1H NMR (CDCl3, 600 MHz) δ 7.13 (1H, d, J = 8.3 Hz), 6.45 (1H, d, J = 2.8 Hz), 6.42 (1H, dd, J = 8.3 Hz, 2.8 Hz), 4.61 (2H, s), 3.98 (2H, t, J = 6.7 Hz), 3.93 (2H, t, J = 6.4 Hz), 3.39 (8H, t, J = 6.9 Hz), 2.24 (1H, brs), 1.83-1.72 (4H, m), 1.61-1.51 (8H, m), 1.49 (4H, m), 1.40-1.19 (52H, m), 0.88 (6H, t, J = 6.9Hz); 13 C NMR (CDCl3, 151 MHz) δ 160.1, 158.1, 129.5, 121.7, 104.4, 99.8, 71.0, 68.1, 68.0, 62.0, 31.9, 29.8, 29.6, 29.5, 29.3, 29.2, 26.2, 26.1, 26.0, 22.7, 14.1.

[0202] (3) 379.6 mg (0.50 mmol) of compound 28 was dissolved in a mixture of 6 mL of THF, 5.7 mL of t-BuOH, and 4.3 mL of water. To the mixture were added 599.9 mg (5.0 mmol) of NaH2PO4, 797.0 μL (7.5 mmol) of 2-methyl-2-butene, and 282.6 mg (2.5 mmol) of NaClO2, and the mixture was stirred at room temperature for 2 hours. After stirring, 20 mL of saturated brine was added to the reaction solution, and the organic layer was extracted three times with 20 mL of ethyl acetate. The organic layer was then washed with 20 mL of saturated aqueous sodium thiosulfate and 20 mL of saturated brine, and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give 394.9 mg (yield quant.) of compound 30. 1H NMR (CDCl3, 600 MHz) δ 10.72 (1H, brs), 8.11 (1H, d, J =8.9 Hz), 6.61 (1H, dd, J = 8.9 Hz, 2.1 Hz), 6.51 (1H, d, J = 2.1 Hz), 4.19 (2H, t, J = 6.9 Hz), 4.00 (2H, t, J = 6.9 Hz), 3.39 (8H, t, J = 6.9 Hz), 1.94-1.84 (2H, m), 1.83-1.75 (2H, m), 1.61-1.52 (8H, m), 1.52-1.41 (4H, m), 1.41-1.19 (52H, m), 0.88 (6H, t, J = 6.9 Hz); 13 C NMR (CDCl3, 151 MHz) δ 165.4, 164.6, 159.0, 135.4, 110.2, 107.0, 99.7, 70.9, 70.9, 70.1, 68.5, 31.9, 29.7, 29.6, 29.5, 29.4, 29.4, 29.3, 29.1, 29.0, 28.8, 26.2, 26.1, 25.9, 25.8, 22.6, 14.1.

[0203] Synthesis of N-(2,4-bis((10-(undecyloxy)decyl)oxy)benzyl)ethanamine (compound 31)

[0204]

[0205] 941.9 mg (1.2 mmol) of compound 28 was dissolved in 17 mL of THF and 8 mL of DMF, and 0.30 g (3.6 mmol) of ethylamine hydrochloride, 0.61 mL (3.6 mmol) of DIPEA, and 0.21 mL (3.6 mmol) of acetic acid were added. Then, 763.0 mg (3.6 mmol) of NaBH(OAc)3 was added and stirred under an argon atmosphere for 1 hour. 15 mL of 1 N hydrochloric acid was added to the stirred reaction solution, and the organic layer was extracted three times with 30 mL of ethyl acetate. This organic layer was washed with 30 mL of saturated brine and dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was crudely purified by column chromatography (chloroform:methanol = 30:1 to 10:1). The organic layer containing compound 31 was washed with 30 mL of saturated sodium bicarbonate water and 30 mL of saturated brine. The organic layer was dried over sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 951.6 mg (yield quant.) of compound 31. 1 H NMR (CDCl3, 600 MHz) δ 7.09 (1H, d, J = 7.6 Hz), 6.45-6.36 (2H, m), 3.98-3.88 (4H, m), 3.71 (2H, s), 3.39 (8H, t, J = 8H, t, J = 6.9 Hz), 2.62 (2H, q, J = 6.9 Hz), 1.85-1.69 (4H, m), 1.62-1.51 (8H, m), 1.50-1.40 (4H, m), 1.39-1.20 (53H, m), 1.10 (3H, t, J = 6.9 Hz), 0.88 (6H, t, J = 6.9 Hz); 13 C NMR (CDCl3, 151 MHz) δ 159.4, 158.1, 130.3, 120.9, 104.2, 99.7, 71.0, 70.9, 68.0, 67.8, 48.9, 43.2, 31.9, 29.8, 29.6, 29.5, 29.5, 29.4, 29.3, 29.3, 26.2, 26.0, 22.7, 15.3, 14.1.

[0206] Synthesis of N-(2,4-bis((10-(undecyloxy)decyl)oxy)benzyl)-1-(2,4-dimethoxyphenyl)methanamine (Compound 32)

[0207]

[0208] 379.7 mg (0.5 mmol) of compound 28 was dissolved in 7 mL of THF, and 0.23 mL (1.5 mmol) of 2,4-dimethoxybenzylamine and 0.09 mL (1.5 mmol) of acetic acid were added. After stirring for 5 minutes, 317.9 mg (1.5 mmol) of NaBH(OAc)3 was added and the mixture was stirred for 1 hour under an argon atmosphere. 20 mL of saturated aqueous sodium bicarbonate was added to the stirred reaction solution, and the organic layer was extracted three times with 30 mL of ethyl acetate. This organic layer was washed with 20 mL of 1 N hydrochloric acid, dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (chloroform:methanol = 20:1) to obtain 466.3 mg of compound 32 (yield 98%). 1 H NMR (CDCl3, 600 MHz) δ 7.23 (1H, d, J = 8.3 Hz), 7.27 (1H, d, J = 8.3 Hz), 6.46-6.37 (4H, m), 4.00-3.86 (8H, m), 3.80-3.73 (6H, m), 3.39 (8H, t, J = 6.9 Hz), 1.79-1.64 (4H, m), 1.61-1.51 (8H, m), 1.47-1.20 (57H, m), 0.88 (6H, t, J = 6.9 Hz); 13 C NMR (CDCl3, 151 MHz) δ 161.5, 160.9, 158.5, 158.0, 132.3, 114.1, 113.7, 104.9, 104.3, 99.6, 98.5, 71.0, 70.9, 68.3, 68.1, 55.4, 55.3, 46.5, 46.2, 31.9, 29.7, 29.6, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 26.2, 26.0, 26.0, 22.7, 14.1.

[0209] Synthesis of 3,4,5-tris((8-(nonyloxy)octyl)oxy)benzaldehyde (Compound 33) and N-(3,4,5-tris((8-(nonyloxy)octyl)oxy)benzyl)ethanamine (Compound 34)

[0210]

[0211] (1) 919.5 mg (1.0 mmol) of compound 3 was dissolved in DCM. The resulting solution was dissolved in 10 mL of DMSO, and 710 μL (10 mmol) and 850 μL (5.0 mmol) of DIPEA were added. To this solution, 477.5 mg (3.0 mmol) of SO3·Py was added in an ice bath and stirred for 1 hour. The reaction was quenched by adding 20 mL of saturated aqueous sodium thiosulfate solution. After adding 10 mL of H2O, the organic layer was extracted twice with 20 mL of DCM. This organic layer was washed with saturated brine, dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue was subjected to column chromatography (hexane:ethyl acetate = 10:1) to obtain 886.6 mg of compound 33 (97% yield).

[0212] (2) 183.5 mg (0.20 mmol) of compound 33 was dissolved in 3.0 mL of THF, and 48.9 mg (0.60 mmol) of ethylamine hydrochloride, 34.3 μL (0.60 mmol) of acetic acid, 102.0 μL (0.60 mmol) of DIPEA, and 2.0 mL of DMF were added and stirred for 1 hour. 127.2 mg (0.60 mmol) of NaBH(OAc)3 was added to this solution and stirred overnight. 10 mL of saturated aqueous sodium bicarbonate was added to this solution to quench the reaction, and the organic layer was extracted twice with 20 mL of ethyl acetate. The organic layer was washed with 10 mL of 1 N hydrochloric acid and 10 mL of saturated brine, dried over sodium sulfate, and the solvent was evaporated under reduced pressure to give 191.3 mg of compound 34 (97% yield). 1H NMR (CDCl3, 600 MHz) δ 6.76 (2H, s), 3.99 (4H, t, J = 6.9 Hz), 3.95-3.86 (4H, m), 3.39 (12H, t, J = 6.9 Hz), 2.82 (2H, q, J = 7.6 Hz), 1.82-1.66 (6H, m), 1.61-1.51 (12H, m), 1.50-1.40 (6H, m), 1.39-1.19 (57H, m), 0.88 (9H, t, J = 7.6 Hz); 13 C NMR (CDCl3, 151 MHz) δ 153.6, 138.6, 125.8, 108.3, 73.5, 71.1, 71.1, 71.1, 69.4, 50.7, 41.0, 32.0, 30.4, 29.9, 29.7, 29.6, 29.5, 29.5, 29.4, 26.4, 26.3, 26.2, 22.8, 14.2, 11.5.

[0213] Synthesis of N-(2,4-dimethoxybenzyl)-1-(3,4,5-tris((8-(nonyloxy)octyl)oxy)phenyl)methanamine (Compound 35)

[0214]

[0215] 183.5 mg (0.20 mmol) of compound 33 was dissolved in 3.0 mL of THF, and 90.4 μL (0.60 mmol) of 2,5-dimethoxybenzylamine and 34.3 μL (0.60 mmol) of acetic acid were added. The mixture was stirred for 40 minutes. 127.2 mg (0.60 mmol) of NaBH(OAc)3 was then added to the solution and stirred for 4 hours. 6.0 mL of saturated aqueous sodium bicarbonate was added to the solution to quench the reaction, and the organic layer was extracted twice with 20 mL of ethyl acetate. The organic layer was washed with 20 mL of 1 N hydrochloric acid and 20 mL of saturated brine, dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue was subjected to column chromatography (chloroform → chloroform:methanol = 20:1 → 10:1) to obtain 205.8 mg of compound 35 (95% yield). 1H NMR (CDCl3, 600 MHz) δ 7.12 (1H, d, J = 8.3 Hz), 6.52 (2H, s), 6.47-6.41 (2H, m), 3.98-3.88 (6H, m), 3.82-3.78 (6H, m), 3.76 (2H, s), 3.68 (2H, s), 3.42-3.33 (12H, m), 3.06 (1H, brs), 1.82-1.68 (6H, m), 1.62-1.51 (12H, m), 1.50-1.40 (6H, m), 1.40-1.20 (54H, m), 0.88 (9H, t, J = 7.6 Hz); 13 C NMR (CDCl3, 151 MHz) δ 160.4, 158.9, 153.2, 137.2, 135.1, 130.9, 120.2, 106.8, 103.8, 98.7, 73.5, 71.1, 71.1, 69.2, 55.5, 55.4, 52.9, 48.3, 32.0, 30.5, 30.0, 29.9, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 26.3, 26.3, 26.2, 26.2, 22.8, 14.2.

[0216] Synthesis of (3,4,5-tris((8-(nonyloxy)octyl)oxy)phenyl)methanamine (Compound 37)

[0217]

[0218] (1) 183.9 mg of compound 3 was dissolved in 2.5 mL of toluene, and 89.6 μL of DBU and 129.0 mg (0.60 mmol) of DPPA were added. oThe mixture was stirred at 37°C for 90 minutes. 10 mL of 1 N HCl was added to the stirred reaction solution, and the organic layer was extracted twice with 20 mL of toluene. This organic layer was washed with 10 mL of saturated sodium bicarbonate water and 10 mL of saturated brine, dried over magnesium sulfate, and evaporated under reduced pressure to obtain compound 36. (2) Next, the residue (compound 36) was dissolved in 5.0 mL of toluene, and 209.8 mg (0.80 mmol) of triphenylphosphine and 72.0 μL (4.0 mmol) of HO were added. The mixture was stirred for 60 minutes. o The mixture was stirred at C for 12 hours. After stirring, the solvent was removed from the solution under reduced pressure, and 20 mL each of hexane and acetonitrile were added. The hexane layer was then extracted twice with 20 mL of hexane. The hexane layer was collected, the solvent was removed under reduced pressure, and the residue was subjected to column chromatography (chloroform:2-propanol = 50:1 → 20:1) to obtain 155.5 mg of compound 37 (yield 85%). 1 H NMR (CDCl3, 600 MHz) δ 6.50 (2H, s), 3.97 (4H, t, J = 6.9 Hz), 3.92 (2H, t, J = 6.9 Hz), 3.78 (2H, s), 3.42-3.35 (12H, m), 1.83-1.69 (6H, m), 1.61-1.52 (12H, m), 1.51-1.41 (8H, m), 1.41-1.20 (54H, m), 0.88 (9H, t, J = 6.9 Hz); 13 C NMR (CDCl3, 151 MHz) δ 153.3, 138.6, 137.1, 105.6, 73.5, 71.1, 71.1, 69.2, 46.9, 32.0, 30.4, 30.0, 29.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 26.3, 26.3, 26.2, 26.2, 22.8, 14.2.

[0219] Synthesis of (2,4,5-tris((8-(nonyloxy)octyl)oxy)phenyl)methanol (compound 39)

[0220]

[0221] (1) 2,4,5-trihydroxybenzaldehyde (493.2 mg, 3.2 mmol) and compound 1 (4.11 g, 12.8 mmol) were dissolved in N-methylpyrrolidone (6.4 mL), potassium carbonate (2.21 g, 16 mmol) was added, and the mixture was heated at 110 o The mixture was stirred at RT for 4 hours. Ethyl acetate (30 mL) was added to the stirred reaction solution, and the potassium carbonate was removed by filtration. The resulting solution was then washed with ethyl acetate (20 mL). The organic layer was washed twice with 8% brine (50 mL) and saturated brine (30 mL), and dried over sodium sulfate. The solvent was removed under reduced pressure, and the residue was added to 2-propanol (60 mL). Compound 38 (1.96 g, 2.14 mmol, 67% yield) was obtained by recrystallization.

[0222] (2) Compound 38 (1.59 g, 1.7 mmol) was dissolved in THF- i This was dissolved in PrOH (20 mL, 7:3), and NaBH4 (96.5 mg, 2.55 mmol) was added. The mixture was stirred at room temperature for 14 hours under an argon atmosphere. Water (20 mL) was added, and the mixture was extracted three times with ethyl acetate (30 mL). The organic layer was washed with saturated brine (20 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane:ethyl acetate = 7:1) to obtain 1.56 g (quant.) of compound 39. 1 H NMR (CDCl3, 600 MHz) δ 6.86 (1H, s), 6.50 (1H, s), 4.60 (2H, d, J = 6.2 Hz), 4.02-3.87 (6H, m), 3.44-3.30 (12H, m), 2.32 (1H, t, J = 6.2 Hz), 1.84-1.71 (6H, m), 1.62-1.17 (72H, m), 0.88 (9H, t, J = 6.9 Hz); 13C NMR (CDCl3, 150 MHz) δ 151.7, 149.7, 143.0, 121.5, 116.8, 100.8, 71.1, 71.0, 71.0, 70.7, 70.0, 68.9, 61.9, 32.0, 29.9, 29.7, 29.6, 29.6, 29.5, 29.5, 29.4, 29.4, 26.3, 26.3, 26.2, 26.1, 22.8, 14.2.

[0223] Synthesis of N-(2,4,5-tris((8-(nonyloxy)octyl)oxy)benzyl)ethanamine (Compound 40)

[0224]

[0225] Compound 38 (2.39 g, 2.6 mmol) was dissolved in THF (20 mL), and ethylamine (2 M in THF 2.6 mL, 5.2 mmol) and acetic acid (300 μL, 5.2 mmol) were added. The mixture was stirred for 40 minutes under an argon atmosphere. o The mixture was stirred at RT for 1 hour. NaBH(OAc)3 (1.1 g, 5.2 mmol) was added to the reaction solution and stirred overnight. The mixture was extracted three times with water (20 mL) and ethyl acetate (20 mL). The organic layer was washed with saturated brine (20 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (dichloromethane:methanol = 30:1) to give compound 40 (1.82 g, 1.92 mmol, 74% yield). 1 H NMR (CDCl3, 600 MHz) δ 7.24 (1H, s), 6.46 (1H, s), 4.07 (2H, s), 4.03-3.89 (6H, m), 3.44-3.34 (12H, m), 2.85 (2H, q, J = 7.6 Hz), 1.99 (0.7H, s), 1.97 (0.3H, s), 1.83-1.69 (6H, m), 1.63-1.50 (12H, m), 1.49-1.20 (63H, m), 0.88 (9H, t, J = 6.9 Hz); 13C NMR (CDCl3, 150 MHz) δ 152.1, 150.8, 143.4, 117.7, 110.2, 99.9, 71.1, 71.1, 71.0, 70.2, 69.7, 69.1, 43.9, 40.5, 32.0, 29.9, 29.7, 29.6, 29.6, 29.5, 29.4, 29.4, 26.3, 26.3, 26.2, 26.1, 22.8, 14.2, 11.3.

[0226] Synthesis of (2,3,4-tris((8-(nonyloxy)octyl)oxy)phenyl)methanol (compound 42)

[0227]

[0228] (1) 2,3,4-trihydroxybenzaldehyde (385.3 mg, 2.5 mmol) and compound 1 (3.94 g, 12.2 mmol) were dissolved in N-methylpyrrolidone (12.5 mL), potassium carbonate (1.52 g, 11 mmol) was added, and the mixture was heated at 90°C under an argon atmosphere. o The mixture was stirred at RT for 18 hours and 30 minutes. The potassium carbonate was removed by filtration and washed with ethyl acetate (30 mL). 5% brine (70 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (30 mL). The organic layer was washed twice with saturated brine (30 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to give compound 41 (2.16 g, 2.36 mmol, 94% yield).

[0229] (2) Compound 41 (917.5 mg, 1.0 mmol) was dissolved in THF- i The product was dissolved in PrOH (10 mL, 7:3) and NaBH (56.7 mg, 1.5 mmol) was added. The mixture was stirred for 1 hour and 30 minutes. Water (20 mL) was added to the reaction solution, which was then extracted three times with hexane (20 mL). The organic layer was washed with saturated brine (20 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure, and the mixture was recrystallized in 2-propanol to give compound 42 (812.0 mg, 0.88 mmol, 88% yield). 11H NMR (CDCl3, 600 MHz) δ 6.93 (1H, d, J = 8.9 Hz), 6.60 (1H, d, J = 8.9 Hz), 4.60 (2H, d, J = 6.2 Hz), 4.10 (2H, t, J = 6.9 Hz), 3.99 - 3.91 (4H, m), 3.39 (12H, t, J = 6.9 Hz), 2.24 (H, t, J = 6.2 Hz), 1.86 - 1.78 (2H, m), 1.78 - 1.71 (4H, m), 1.70 - 1.62 (2H, m), 1.62 - 1.52 (12H, m), 1.51 - 1.20 (58H, m), 0.88 (9H, t, J = 6.9 Hz); 13 13C NMR (CDCl3, 150 MHz) δ 153.5, 151.6, 141.6, 126.9, 123.2, 108.0, 74.1, 73.7, 71.2, 71.1, 71.1, 68.8, 62.1, 32.0, 30.6, <30.5, 29.9, 29.9, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 26.3, 26.3, 26.2, 26.2, 26.2, 22.8, 14.3.

[0230] Synthesis of N-(2,3,4-tris((8-(nonyloxy)octyl)oxy)benzyl)ethanamine (Compound 43)

[0231]

[0232] Dissolve Compound 41 (917.5 mg, 1.0 mmol) in THF (20 mL), add ethylamine (2 M in THF 1.0 mL, 2.0 mmol) and acetic acid (115 μL, 2.0 mmol), and under an argon atmosphere, 40 o It should be noted that there seems to be a small error in the original text where "29.5" in the 13C NMR data is written as "<30.5" in the translation. It should be "29.5" in both the original and the translation for accuracy.The mixture was stirred at RT for 1 hour. NaBH(OAc)3 (423 mg, 2.0 mmol) was added to the reaction solution and stirred overnight. Water (20 mL) and saturated aqueous sodium bicarbonate (20 mL) were added, and the mixture was extracted three times with ethyl acetate (20 mL). The organic layer was washed with saturated brine (20 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (dichloromethane:methanol = 30:1) to give compound 43 (513.7 mg, 0.54 mmol, 54% yield). 1 H NMR (CDCl3, 600 MHz) δ 6.91 (1H, d, J = 8.3 Hz), 6.58 (1H, d, J = 8.3 Hz), 4.04 (2H, t, J = 6.9 Hz), 3.99-3.89 (4H, m), 3.73 (2H, s), 3.39 (12H, t, J = 6.9 Hz), 2.66 (2H, q, J = 6.9 Hz), 2.11 (1H, brs), 1.86-1.69 (6H, m), 1.62-1.51 (12H, m), 1.51-1.41 (6H, m), 1.40-1.20 (54H, m), 1.14 (3H, t, J = 6.9 Hz), 0.88 (9H, t, J = 6.9 Hz); 13 C NMR (CDCl3, 150 MHz) δ 152.9, 151.7, 141.7, 125.5, 124.1, 108.0, 73.9, 73.6, 71.1, 71.1, 71.1, 68.8, 48.9, 43.4, 32.0, 31.1, 30.7, 30.5, 29.9, 29.7, 29.7, 29.6, 29.5, 29.4, 26.3, 26.2, 26.2, 22.8, 15.1, 14.3.

[0233] Synthesis of (2,4,6-tris((8-(nonyloxy)octyl)oxy)phenyl)methanol (compound 45)

[0234]

[0235] (1) 2,4,6-trihydroxybenzaldehyde (385.3 mg, 2.5 mmol) and compound 1 (3.94 g, 12.2 mmol) were dissolved in N-methylpyrrolidone (12.5 mL), potassium carbonate (1.52 g, 11 mmol) was added, and the mixture was heated at 90°C under an argon atmosphere. o The mixture was stirred at RT for 17 hours and 30 minutes at RT. The potassium carbonate was removed by filtration and washed with ethyl acetate (50 mL). The organic layer was washed twice with 13% brine (60 mL) and saturated brine (30 mL), and dried over sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane:ethyl acetate=10:1) to give compound 44 (1.87 g, 2.03 mmol, 82% yield).

[0236] (2) Compound 44 (917.5 mg, 1.0 mmol) was dissolved in THF- i The residue was dissolved in PrOH (10 mL, 7:3), and NaBH4 (85.6 mg, 2.0 mmol) was added. The mixture was stirred for 1 hour and 30 minutes. Water (20 mL) was added, and the mixture was extracted three times with hexane (30 mL). The organic layer was washed with saturated brine (20 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane:ethyl acetate = 10:1) to give compound 45 (905.6 mg, 0.99 mmol, 99% yield). 1 H NMR (CDCl3, 600 MHz) δ 6.09 (2H, s), 4.72 (2H, d, J =6.9 Hz), 4.01-3.86 (6H, m), 3.39 (12H, m), 2.41 (1H, t, J = 6.9 Hz), 1.85-1.71 (6H, m), 1.60-1.51 (12H, m), 1.50-1.20 (60H, m), 0.88 (9H, t, J = 6.9 Hz); 13C NMR (CDCl3, 150 MHz) δ 160.4, 158.6, 110.3, 91.9, 71.1, 71.0, 68.5, 68.1, 54.9, 32.0, 29.9, 29.8, 29.7, 29.6, 29.5, 29.5, 29.4, 29.4, 29.4, 26.3, 26.2, 26.2, 26.1, 22.8, 14.2.

[0237] Synthesis of H-Leu-O-TagA (Compound 46)

[0238]

[0239] Compound 3 (459.8 mg, 0.50 mmol) was dissolved in ethyl acetate (10 mL). Fmoc-Leu-OH (265.1 mg, 0.75 mmol), DMAP (12.2 mg, 0.10 mmol), and DIPCI (115.4 μL, 0.75 mmol) were added and the mixture was stirred for 4 hours. 1-Methylpiperazine (27.5 μL, 0.25 mmol) was added to the reaction solution and stirred for 10 minutes. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and the mixture was stirred for 10 minutes. 6 M hydrochloric acid (3.75 mL), saturated brine (30 mL), and acetonitrile (10 mL) were then added, followed by extraction three times with ethyl acetate (30 mL). The organic layer was washed with 1 M hydrochloric acid (3.0 mL) and saturated brine (30 mL), saturated brine (30 mL), saturated sodium bicarbonate solution (30 mL), and saturated brine (30 mL), dried over magnesium sulfate, and the solvent was evaporated under reduced pressure to give H-Leu-O-TagA (compound 46).

[0240] Synthesis of protected leucine enkephalin (compound 47)

[0241]

[0242] H-Leu-O-TagA (compound 46) was dissolved in ethyl acetate (5.0 mL), and Fmoc-Phe-OH (251.8 mg, 0.65 mmol), DIPEA (281 μL, 1.65 mmol), and COMU (278.4 mg, 0.65 mmol) were added and stirred for 30 min. 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and stirred for 10 min. A mixture of 6 M hydrochloric acid (3.75 mL) and saturated brine (30 mL) was then added, and the mixture was extracted three times with ethyl acetate (30 mL). The organic layer was washed with a mixture of 1 M hydrochloric acid (3.0 mL) and saturated brine (30 mL), saturated brine (30 mL), saturated aqueous sodium bicarbonate (30 mL), and saturated brine (30 mL), and then dried over magnesium sulfate. The solvent was removed under reduced pressure to obtain H-Phe-Leu-O-TagA. The above procedure was then repeated using the appropriate Fmoc-AA-OH or Boc-Tyr(tBu)-OH to obtain 541.5 mg (67% yield) of the protected leucine enkephalin (compound 47). Exact Mass [M+H]+ calculated for [C95H162N5O15]+ 1613.2, observed 1613.7. HPLC analysis conditions were as follows (Figure 1). Solvent A: THF, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 70:30 → 95:5 (15 min) Column: YMC-Triart C18 150 x 2.1 mm, S-1.9 μm, 12 nm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0243] Synthesis of leucine enkephalin (compound 48)

[0244]

[0245] Protected leucine enkephalin (compound 47) (82.3 mg, 0.050 mmol) was dissolved in a TFA-triisopropylsilane-HO mixed solution (95:2.5:2.5, 2.0 mL) and stirred for 6 hours. 30 mL of diisopropyl ether was added to the solution, and the mixture was centrifuged in a cool place. The supernatant was removed, and the residue was dried to obtain 47.6 mg (quantity) of leucine enkephalin (compound 48). Exact mass [M+H]+ calculated for [C28H38N5O7]+ 556.3, observed 556.3. HPLC analysis conditions were as follows (Figure 2). Solvent A: MeCN, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 15:85 → 15:85 (5 min) → 57.5:42.5 (25 min) Column: YMC-Triart C18 150 x 2.1 mm, S-1.9 μm, 12 nm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0246] Synthesis of H-Leu-O-TagA (Compound 46)

[0247]

[0248] Compound 3 (275.9 mg, 0.30 mmol) was dissolved in anisole (6.0 mL). Fmoc-Leu-OH (159.0 mg, 0.45 mmol), DMAP (7.3 mg, 0.060 mmol), and DIPCI (69.3 μL, 0.45 mmol) were added and the mixture was stirred for 50 min. 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (660 μL, 6.0 mmol) and DBU (313 μL, 2.1 mmol) were added and the mixture was stirred for 10 min. A mixture of 6 M hydrochloric acid (2.25 mL) and saturated brine (30 mL) was then added, and the mixture was extracted three times with ethyl acetate (20 mL). The organic layer was washed with 1 M hydrochloric acid (1.8 mL) and saturated brine (30 mL), saturated brine (30 mL), saturated aqueous sodium bicarbonate (30 mL), and saturated brine (30 mL), dried over magnesium sulfate, and the solvent was evaporated under reduced pressure to give H-Leu-O-TagA (compound 46).

[0249] Synthesis of Fmoc-Phe-Leu-O-TagA (Compound 49)

[0250]

[0251] The H-Leu-O-TagA (compound 46) obtained above was dissolved in anisole (10.0 mL). Fmoc-Phe-OH (151.1 mg, 0.39 mmol), DIPEA (168.4 μL, 0.99 mmol), and COMU (167.0 mg, 0.39 mmol) were added and the mixture was stirred for 16.5 hours. 1 M hydrochloric acid (20 mL) was added, and the mixture was extracted twice with ethyl acetate (20 mL). The organic layer was washed with saturated aqueous sodium bicarbonate (20 mL) and saturated brine (30 mL) and dried over magnesium sulfate. The solvent was removed under reduced pressure, and the residue was subjected to column chromatography (c-Hex: EtOAc = 7:1 → 4:1) to obtain 394.6 mg of Fmoc-Phe-Leu-O-TagA (compound 49) (94% yield). 1H NMR (CDCl3, 400MHz) δ7.76 (2H, d, J = 7.3 Hz), 7.60-7.49 (2H, m), 7.40 (2H, t, J = 7.3 Hz), 7.35-7.09 (7H, m), 6.51 (2H, s), 6.15 (1H, d, J = 6.0 Hz), 5.32 (1H, d, J = 7.3 Hz), 5.03 (2H, s), 4.65-4.53 (1H, m), 4.51-4.38 (2H, m), 4.38-4.27 (1H, m), 4.19 (1H, t, J = 6.9 Hz), 4.01-3.81 (6H, m), 3.38 (12H, t, J = 6.9 Hz), 3.18-2.84 (2H, m), 1.84-1.67 (6H, m), 1.63-1.13 (75H, m), 0.96-0.76 (15H, m); 13 C NMR (CDCl3, 100 MHz) δ 172.3, 170.5, 156.0, 153.3, 143.8, 143.8, 141.4, 138.4, 136.3, 130.3, 129.5, 128.8, 127.8, 127.2, 125.2, 125.1, 120.1, 107.0, 73.5, 71.1, 71.1, 71.0, 69.2, 67.5, 67.2, 56.0, 56.0, 51.1, 47.2, 41.6, 38.4, 32.0, 30.4, 29.9, 29.7, 29.6, 29.6, 29.5, 29.4, 26.3, 26.3, 26.2, 26.1, 24.8, 22.8, 22.0, 14.2.

[0252] Synthesis of H-Leu-O-TagA (compound 46)

[0253]

[0254] Compound 3 (275.9 mg, 0.30 mmol) was dissolved in isopropyl acetate (3.0 mL). Fmoc-Leu-OH (159.0 mg, 0.45 mmol), DMAP (7.3 mg, 0.060 mmol), and DIPCI (69.3 μL, 0.45 mmol) were added and stirred for 30 min. Isopropyl acetate (3.0 mL) and 1-methylpiperazine (24.8 μL, 0.23 mmol) were added to the reaction solution and stirred for 10 min. 1-methylpiperazine (660 μL, 6.0 mmol) and DBU (313 μL, 2.1 mmol) were added and stirred for 10 min. A mixture of 6 M hydrochloric acid (2.25 mL), saturated brine (30 mL), and acetonitrile (5.0 mL) was then added, followed by extraction three times with ethyl acetate (20 mL). The organic layer was washed with a mixture of 1 M hydrochloric acid (1.8 mL) and saturated brine (20 mL), saturated brine (30 mL), saturated aqueous sodium bicarbonate (20 mL), and saturated brine (30 mL), dried over magnesium sulfate, and the solvent was evaporated under reduced pressure to give H-Leu-O-TagA (compound 46).

[0255] Synthesis of Fmoc-Phe-Leu-O-TagA (Compound 49)

[0256]

[0257] The H-Leu-O-TagA (compound 46) obtained above was dissolved in isopropyl acetate (6.0 mL). Fmoc-Phe-OH (151.1 mg, 0.39 mmol), DIPEA (168.4 μL, 0.99 mmol), and COMU (167.0 mg, 0.39 mmol) were added and the mixture was stirred for 2.5 hours. 1 M hydrochloric acid (20 mL) was added, followed by extraction twice with ethyl acetate (20 mL). The organic layer was washed with saturated aqueous sodium bicarbonate (20 mL) and saturated brine (30 mL) and dried over magnesium sulfate. The solvent was removed under reduced pressure, and the residue was subjected to column chromatography (c-Hex: EtOAc = 7:1 → 4:1) to obtain 241.4 mg of Fmoc-Phe-Leu-O-TagA (compound 49) (57% yield).

[0258] Synthesis of H-Leu-O-TagA (Compound 46)

[0259]

[0260] Compound 3 (275.9 mg, 0.30 mmol) was dissolved in butyl acetate (3.0 mL). Fmoc-Leu-OH (159.0 mg, 0.45 mmol), DMAP (7.3 mg, 0.060 mmol), and DIPCI (69.3 μL, 0.45 mmol) were added and stirred for 20 min. To the reaction solution, butyl acetate (3.0 mL) and 1-methylpiperazine (24.8 μL, 0.23 mmol) were added and stirred for 10 min. 1-methylpiperazine (660 μL, 6.0 mmol) and DBU (313 μL, 2.1 mmol) were added and stirred for 10 min. A mixture of 6 M hydrochloric acid (2.25 mL), saturated brine (30 mL), and acetonitrile (5.0 mL) was then added, followed by extraction three times with ethyl acetate (20 mL). The organic layer was washed with a mixture of 1 M hydrochloric acid (1.8 mL) and saturated brine (20 mL), saturated brine (30 mL), saturated aqueous sodium bicarbonate (20 mL), and saturated brine (30 mL), dried over magnesium sulfate, and the solvent was evaporated under reduced pressure to give H-Leu-O-TagA (compound 46).

[0261] Synthesis of Fmoc-Phe-Leu-O-TagA (Compound 49)

[0262]

[0263] The H-Leu-O-TagA (compound 46) obtained above was dissolved in butyl acetate (6.0 mL). Fmoc-Phe-OH (151.1 mg, 0.39 mmol), DIPEA (168.4 μL, 0.99 mmol), and COMU (167.0 mg, 0.39 mmol) were added and the mixture was stirred for 17.5 hours. 1 M hydrochloric acid (20 mL) was added, and the mixture was extracted three times with ethyl acetate (20 mL). The organic layer was washed with saturated aqueous sodium bicarbonate (20 mL) and saturated brine (30 mL) and dried over magnesium sulfate. The solvent was removed under reduced pressure, and the residue was subjected to column chromatography (c-Hex: EtOAc = 7:1 → 4:1) to obtain 385.6 mg of Fmoc-Phe-Leu-O-TagA (compound 49) (92% yield).

[0264] Synthesis of H-Leu-O-TagB (Compound 50)

[0265]

[0266] Compound 39 (459.8 mg, 0.50 mmol) was dissolved in ethyl acetate (10 mL). Fmoc-Leu-OH (265.1 mg, 0.75 mmol), DMAP (12.2 mg, 0.10 mmol), and DIPCI (115.4 μL, 0.75 mmol) were added and stirred for 15 min. 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and stirred for 10 min. 0.7 M hydrochloric acid (32 mL) was then added, and the mixture was extracted three times with ethyl acetate (20 mL). The mixture was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give H-Leu-O-TagB (compound 50).

[0267] Synthesis of protected leucine enkephalin (compound 51)

[0268]

[0269] H-Leu-O-TagB (compound 50) was dissolved in ethyl acetate (10 mL), and Fmoc-Phe-OH (251.8 mg, 0.65 mmol), DIPEA (281 μL, 1.65 mmol), and COMU (278.4 mg, 0.65 mmol) were added and stirred for 30 min. 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and stirred for 10 min. A mixture of 6 M hydrochloric acid (3.75 mL) and saturated brine (30 mL) was then added, and the mixture was extracted three times with ethyl acetate (30 mL). The organic layer was washed with a mixture of 1 M hydrochloric acid (3.0 mL) and saturated brine (30 mL), saturated brine (30 mL), saturated aqueous sodium bicarbonate (30 mL), and saturated brine (30 mL), and then dried over magnesium sulfate. The solvent was removed under reduced pressure to obtain H-Phe-Leu-O-TagB. The above procedure was then repeated using the appropriate Fmoc-AA-OH or Boc-Tyr(tBu)-OH to obtain 1.28 g of crude product containing the protected leucine enkephalin (compound 51). Exact Mass [M+H]+ calculated for [C95H162N5O15]+ 1613.2, observed 1613.7. HPLC analysis conditions were as follows (Figure 3). Solvent A: THF, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 70:30 → 95:5 (15 min) Column: Nomura Chemical Develosil C8 150 x 2.0 mm, 3 μm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0270] Synthesis of leucine enkephalin (compound 48)

[0271]

[0272] Protected leucine enkephalin (compound 51) (32.3 mg, 0.020 mmol) was dissolved in TFA-TIS-HO (5.0 mL, 95:2.5:2.5) and stirred overnight. The solvent was removed under reduced pressure, diisopropyl ether (10 mL) was added, and the mixture was centrifuged in a refrigerator to obtain leucine enkephalin (compound 48) (8.0 mg, 0.014 mmol, 70% yield). Exact mass [M+H]+ calculated for [C28H38N5O7]+ 556.3, observed 556.3. HPLC analysis conditions were as follows (Figure 4). Solvent A: MeCN, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 15:85 → 15:85 (5 min) → 57.5:42.5 (25 min) Column: Nomura Chemical Develosil C8 150 x 2.0 mm, 3 μm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0273] Synthesis of H-Leu-O-TagC (Compound 52)

[0274]

[0275] Compound 42 (459.8 mg, 0.50 mmol) was dissolved in ethyl acetate (10 mL). Fmoc-Leu-OH (265.1 mg, 0.75 mmol), DMAP (12.2 mg, 0.10 mmol), and DIPCI (115.4 μL, 0.75 mmol) were added and stirred for 15 min. 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and stirred for 10 min. 0.7 M hydrochloric acid (32 mL) was then added, and the mixture was extracted three times with ethyl acetate (20 mL). The mixture was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give H-Leu-O-TagC (compound 52).

[0276] Synthesis of protected leucine enkephalin (compound 53)

[0277]

[0278] H-Leu-O-TagC (compound 52) was dissolved in ethyl acetate (10 mL), and Fmoc-Phe-OH (251.8 mg, 0.65 mmol), DIPEA (281 μL, 1.65 mmol), and COMU (278.4 mg, 0.65 mmol) were added and stirred for 30 min. 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and stirred for 10 min. A mixture of 6 M hydrochloric acid (3.75 mL) and saturated brine (30 mL) was then added, and the mixture was extracted three times with ethyl acetate (30 mL). The organic layer was washed with a mixture of 1 M hydrochloric acid (3.0 mL) and saturated brine (30 mL), saturated brine (30 mL), saturated aqueous sodium bicarbonate (30 mL), and saturated brine (30 mL), and then dried over magnesium sulfate. The solvent was removed under reduced pressure to obtain H-Phe-Leu-O-TagC. The above procedure was then repeated using the appropriate Fmoc-AA-OH or Boc-Tyr(tBu)-OH to obtain 966.6 mg of crude product containing the protected leucine enkephalin (compound 53). Exact Mass [M+H]+ calculated for [C95H162N5O15]+ 1613.2, observed 1613.4. HPLC analysis conditions were as follows (Figure 5). Solvent A: THF, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 70:30 → 95:5 (15 min) Column: Nomura Chemical Develosil C8 150 x 2.0 mm, 3 μm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0279] Synthesis of leucine enkephalin (compound 48)

[0280]

[0281] Protected leucine enkephalin (compound 53) (32.3 mg, 0.020 mmol) was dissolved in TFA-TIS-HO (5.0 mL, 95:2.5:2.5) and stirred for 2 hours. The solvent was removed under reduced pressure, diisopropyl ether (20 mL) was added, and the mixture was filtered to obtain leucine enkephalin (compound 48) (6.0 mg, 0.011 mmol, 54% yield). Exact mass [M+H]+ calculated for [C28H38N5O7]+ 556.3, observed 556.2. HPLC analysis conditions were as follows (Figure 6). Solvent A: MeCN, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 15:85 → 15:85 (5 min) → 57.5:42.5 (25 min) Column: Nomura Chemical Develosil C8 150 x 2.0 mm, 3 μm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0282] Synthesis of H-Pro-NEt-TagD (Compound 54)

[0283]

[0284] 236.6 mg (0.30 mmol) of compound 31, 139.3 mg (0.39 mmol) of Fmoc-Pro-OH, and 128.0 μL (0.99 mmol) of DIPEA were dissolved in 10 mL of THF, followed by the addition of 167.0 mg (0.39 mmol) of COMU and stirring for 20 minutes. 15.4 μL (0.14 mmol) of 1-methylpiperazine was added to the reaction solution and stirred for 10 minutes. 660 μL (6.0 mmol) of 1-methylpiperazine and 313 μL (2.1 mmol) of DBU were added and stirred for 10 minutes. 2.25 mL of 6 N HCl and 40 mL of HO were then added. The mixture was extracted three times with 40 mL of cyclohexane-THF (1:1). The organic layer was washed with 30 mL of saturated aqueous sodium bicarbonate and 15% brine, dried over magnesium sulfate, and then the solvent was evaporated under reduced pressure to obtain H-Pro-NEt-TagD (compound 54).

[0285] Synthesis of protected leuprorelin (compound 55)

[0286]

[0287] H-Pro-NEt-Tag (compound 54) was dissolved in THF (10 mL), and Fmoc-Arg(Pbf)-OH (253.0 mg, 0.39 mmol), DIPEA (128.0 μL, 0.99 mmol), and COMU (167.0 mg, 0.39 mmol) were added and stirred for 30 min. 1-Methylpiperazine (15.4 μL, 0.14 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (660 μL, 6.0 mmol) and DBU (313 μL, 2.1 mmol) were added and stirred for 10 min. 0.3 M HCl (40 mL) was then added, and the mixture was extracted three times with THF-c-Hex (1:1, 40 mL). The organic layer was washed once with saturated aqueous sodium bicarbonate (40 mL) and twice with 13% brine (40 mL), then dried over magnesium sulfate. The solvent was removed under reduced pressure to give H-Arg(PbF)-Pro-NEt-TagD. The above procedure was then repeated using the appropriate Fmoc-AA-OH or pGlu-OH to give 482.4 mg (60% yield) of the protected leuprorelin (compound 55). Exact Mass [M+H]+ calculated for [C 153 H 229 N 16 O 21 S]+ 2658.7, observed 2658.4. The HPLC measurement conditions were as follows (Figure 7). Solvent A: THF, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 70:30 → 90:10 (35 min) Column: Nomura Chemical ODS-UG-5, inner diameter 2.0 mm, length 150 mm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0288] Synthesis of leuprorelin (compound 56)

[0289]

[0290] 26.6 mg of protected leuprorelin (compound 55) was dissolved in 2.0 mL of TFA-MQ water-triisopropylsilane (1.90:0.05:0.05) and stirred for 3 hours. 10 mL of MQ water was added to the reaction solution, followed by filtration through a PTFE filter. The filtrate was evaporated under reduced pressure, and 20 mL of diisopropyl ether was added to the residue. After refrigerated centrifugation, the supernatant was removed, 20 mL of diisopropyl ether was added, and the mixture was refrigerated and centrifuged again to remove the supernatant. Finally, the solvent was evaporated under reduced pressure by lyophilization, yielding 10.4 mg of leuprorelin (compound 56) (86% yield, 82% HPLC purity). Exact mass [M+H]+ calculated for [C59H85N16O12]+ 1209.7, observed 1209.7. HPLC analysis conditions were as follows (Figure 8). Solvent A: MeCN, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 15:85 → 15:85 (5 min) → 42.5:57.5 (35 min) Column: Nomura Chemical ODS-UG-5, inner diameter 2.0 mm, length 150 mm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0291] Synthesis of H-Pro-NEt-TagB (Compound 57)

[0292]

[0293] Compound 40 (1.89 g, 2.0 mmol) was dissolved in THF-DMF (10 mL, 9:1). Fmoc-Pro-OH (877.2 mg, 2.6 mmol), DIPEA (1.12 mL, 6.6 mmol), and COMU (1.11 g, 2.6 mmol) were added and stirred for 15 min. 1-Methylpiperazine (66 μL, 0.60 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (1.49 mL, 10 mmol) were added and stirred for 10 min. 0.9 M hydrochloric acid (32.5 mL) was added, and the mixture was extracted three times with THF-cyclohexane (20 mL, 4:1). The organic layer was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL) and dried over sodium sulfate. The solvent was evaporated under reduced pressure to obtain H-Pro-NEt-TagB (compound 57).

[0294] Synthesis of protected leuprorelin (compound 58)

[0295]

[0296] H-Pro-NEt-TagB (compound 57) was dissolved in THF-DMF (10 mL, 9:1). Fmoc-Arg(Pbf)-OH (1.69 g, 2.6 mmol), DIPEA (1.12 mL, 6.6 mmol), and COMU (1.11 g, 2.6 mmol) were added and stirred for 15 min. 1-Methylpiperazine (66 μL, 0.60 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (1.49 mL, 10 mmol) were added and stirred for 10 min. 0.9 M hydrochloric acid (32.5 mL) was added, and the mixture was extracted three times with THF-cyclohexane (20 mL, 4:1). The organic layer was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure to give H-Arg(Pbf)-Pro-NEt-TagB. The above procedure was then repeated using the appropriate Fmoc-AA-OH or Boc-pGlu-OH to give 1.85 g (31% yield) of the protected leuprorelin (compound 58). Exact Mass [M+2H]2+ calculated for [C 167 H 256 N 16 O 25 [S]2+ 1459.0, observed 1459.5. The HPLC measurement conditions were as follows (Figure 9): Solvent A: THF, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 70:30 → 95:5 (15 min) Column: YMC-Triart C18 150 x 2.1 mm, S-1.9 μm, 12 nm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0297] Synthesis of leuprorelin (compound 56)

[0298]

[0299] Protected leuprorelin (compound 58) (58.4 mg, 0.020 mmol) was dissolved in TFA-TIS-HO (95:2.5:2.5) and stirred for 2 hours. Diisopropyl ether (20 mL) was added and the mixture was cooled on ice for 5 minutes. Leuprorelin (compound 56) (11.5 mg, 0.0095 mmol, 48% yield) was obtained by filtration. Exact mass [M+H]+ calculated for [C59H85N16O12]+ 1209.7, observed 1209.4. HPLC analysis conditions were as follows (Figure 10). Solvent A: MeCN, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 70:30 → 95:5 (15 min) Column: 15:85 → 15:85 (5 min) → 57.5:42.5 (25 min) Flow rate: 0.20 mL / min Column temperature: 40 o C

[0300] Synthesis of H-Pro-NEt-TagC (Compound 59)

[0301]

[0302] Compound 43 (473.3 mg, 0.50 mmol) was dissolved in ethyl acetate (10 mL). Fmoc-Pro-OH (219.3 mg, 0.65 mmol), DIPEA (280 μL, 1.65 mmol), and COMU (278.4 mg, 0.65 mmol) were added and stirred for 15 minutes. 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 minutes. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (552 μL, 3.5 mmol) were added and stirred for 10 minutes. 0.7 M hydrochloric acid (32 mL) was added, and the mixture was extracted three times with ethyl acetate (20 mL). The organic layer was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL) and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give H-Pro-NEt-TagC (compound 59).

[0303] Synthesis of protected leuprorelin (compound 60)

[0304]

[0305] H-Pro-NEt-TagC (compound 59) was dissolved in ethyl acetate (10 mL). Fmoc-Arg(Pbf)-OH (421.7 mg, 0.65 mmol), DIPEA (280 μL, 1.65 mmol), and COMU (278.4 mg, 0.65 mmol) were added and stirred for 15 min. 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (552 μL, 3.5 mmol) were added and stirred for 10 min. 0.7 M hydrochloric acid (32 mL) was added, and the mixture was extracted three times with ethyl acetate (20 mL). The organic layer was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure to give H-Arg(Pbf)-Pro-NEt-TagC. The above procedure was then repeated using the appropriate Fmoc-AA-OH or Boc-pGlu-OH to give 750.9 mg (51% yield) of the protected leuprorelin (compound 60). Exact Mass [M+2H] 2+ calculated for [C 167 H 256 N 16 O 25 S] 2+ 1459.0, observed 1459.8. The HPLC measurement conditions were as follows (FIG. 11): Solvent A: THF, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 70:30 → 95:5 (15 min) Column: Nomura Chemical Develosil C8 150 x 2.0 mm, 3 μm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0306] Synthesis of leuprorelin (compound 56)

[0307]

[0308] Protected leuprorelin (compound 60) (58.4 mg, 0.020 mmol) was dissolved in TFA-TIS-HO (95:2.5:2.5) and stirred at 40°C for 2 hours. Diisopropyl ether (20 mL) was added and the mixture was cooled on ice for 5 minutes. 20.8 mg (0.0172 mmol, 86% yield) of leuprorelin (compound 56) was obtained by filtration. Exact Mass [M+H] + calculated for [C 59 H 85 N 16 O 12 ] + 1209.7, observed 1209.3. The HPLC measurement conditions were as follows (FIG. 12): Solvent A: MeCN, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 70:30 → 95:5 (15 min) Column: 15:85 → 15:85 (5 min) → 57.5:42.5 (25 min) Flow rate: 0.20 mL / min Column temperature: 40 o C

[0309] Synthesis of H-Ala-O-TagA (Compound 61)

[0310]

[0311] Compound 3 (459.8 mg, 0.50 mmol) was dissolved in toluene (5 mL). Fmoc-Ala-OH (233.5 mg, 0.75 mmol), DMAP (12.2 mg, 0.10 mmol), and DIPCI (115.4 μL, 0.75 mmol) were added and stirred for 15 min. 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and stirred for 10 min. 0.7 M hydrochloric acid (32 mL) was then added, and the mixture was extracted three times with THF-cyclohexane (20 mL, 4:1). The mixture was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give H-Ala-O-Tag (Compound 61).

[0312] Synthesis of Fmoc-Ala-Ala-Ala-Ala-O-TagA (Compound 62)

[0313]

[0314] H-Ala-O-TagA (compound 61) was dissolved in THF-DMF (10 mL, 9:1) and stirred for 15 min with Fmoc-Ala-OH·HO (214.1 mg, 0.65 mmol), DIPEA (280 μL, 1.65 mmol), and COMU (278.4 mg, 0.65 mmol). 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and stirred for 10 min. 0.7 M hydrochloric acid (32 mL) was then added, and the mixture was extracted three times with THF-cyclohexane (20 mL, 4:1). The mixture was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL), and dried over sodium sulfate. The solvent was removed under reduced pressure to give H-Ala-Ala-O-TagA. The above procedure was then repeated using Fmoc-Ala-OH to give 354.2 mg (0.25 mmol, 50% yield) of Fmoc-Ala-Ala-Ala-O-TagA (Compound 62). Exact Mass [M+H] + calculated for [C 85 H 141 N4O 13 ] + 1426.1, observed 1426.6. The HPLC measurement conditions were as follows (FIG. 13): Solvent A: THF, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 70:30 → 95:5 (15 min) Column: Nomura Chemical Develosil C8 150 x 2.0 mm, 3 μm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0315] H-Ala-O-TagB (compound 63)

[0316]

[0317] Compound 39 (459.8 mg, 0.50 mmol) was dissolved in toluene (5 mL). Fmoc-Ala-OH (233.5 mg, 0.75 mmol), DMAP (12.2 mg, 0.10 mmol), and DIPCI (115.4 μL, 0.75 mmol) were added and stirred for 15 min. 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and stirred for 10 min. 0.7 M hydrochloric acid (32 mL) was then added, and the mixture was extracted three times with THF-cyclohexane (20 mL, 4:1). The mixture was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give H-Ala-O-Tag (Compound 63).

[0318] Synthesis of Fmoc-Ala-Ala-Ala-Ala-O-TagB (Compound 64)

[0319]

[0320] H-Ala-O-TagB (compound 63) was dissolved in THF-DMF (10 mL, 9:1) and stirred for 15 min with Fmoc-Ala-OH·HO (214.1 mg, 0.65 mmol), DIPEA (280 μL, 1.65 mmol), and COMU (278.4 mg, 0.65 mmol). 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and stirred for 10 min. 0.7 M hydrochloric acid (32 mL) was then added, and the mixture was extracted three times with THF-cyclohexane (20 mL, 4:1). The mixture was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL), and dried over sodium sulfate. The solvent was removed under reduced pressure to give H-Ala-Ala-O-TagB. The above procedure was then repeated using Fmoc-Ala-OH to give 438.3 mg (0.31 mmol, 62%) of Fmoc-Ala-Ala-Ala-O-TagB (Compound 64). Exact Mass [M+H] + calculated for [C 85 H 141 N4O 13 ] + 1426.1, observed 1426.8. The HPLC measurement conditions were as follows (FIG. 14): Solvent A: THF, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 70:30 → 95:5 (15 min) Column: Nomura Chemical Develosil C8 150 x 2.0 mm, 3 μm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0321] H-Ala-O-TagC (compound 65)

[0322]

[0323] Compound 42 (459.8 mg, 0.50 mmol) was dissolved in toluene (5 mL). Fmoc-Ala-OH (233.5 mg, 0.75 mmol), DMAP (12.2 mg, 0.10 mmol), and DIPCI (115.4 μL, 0.75 mmol) were added and stirred for 15 min. 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and stirred for 10 min. 0.7 M hydrochloric acid (32 mL) was then added, and the mixture was extracted three times with THF-cyclohexane (20 mL, 4:1). The mixture was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give H-Ala-O-TagC (Compound 65).

[0324] Synthesis of Fmoc-Ala-Ala-Ala-Ala-O-TagC (Compound 66)

[0325]

[0326] H-Ala-O-TagC (compound 65) was dissolved in THF-DMF (10 mL, 9:1) and stirred for 15 min with Fmoc-Ala-OH·HO (214.1 mg, 0.65 mmol), DIPEA (280 μL, 1.65 mmol), and COMU (278.4 mg, 0.65 mmol). 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and stirred for 10 min. 0.7 M hydrochloric acid (32 mL) was then added, and the mixture was extracted three times with THF-cyclohexane (20 mL, 4:1). The mixture was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give H-Ala-Ala-O-TagC. The above procedure was then repeated using Fmoc-Ala-OH to give 867.3 g (quantity) of Fmoc-Ala-Ala-Ala-Ala-O-TagC (compound 66). Exact Mass [M+H] + calculated for [C 85 H 141 N4O 13 ]+ 1426.1, observed 1426.6. The HPLC measurement conditions were as follows (Figure 15). Solvent A: THF, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 70:30 → 95:5 (15 min) Column: Nomura Chemical Develosil C8 150 x 2.0 mm, 3 μm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0327] H-Ala-O-TagE (compound 67)

[0328]

[0329] Compound 45 (459.8 mg, 0.50 mmol) was dissolved in toluene (5 mL). Fmoc-Ala-OH (233.5 mg, 0.75 mmol), DMAP (12.2 mg, 0.10 mmol), and DIPCI (115.4 μL, 0.75 mmol) were added and stirred for 15 min. 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and stirred for 10 min. 0.7 M hydrochloric acid (32 mL) was then added, and the mixture was extracted three times with THF-cyclohexane (20 mL, 4:1). The mixture was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give H-Ala-O-TagE (compound 67).

[0330] Synthesis of Fmoc-Ala-Ala-Ala-Ala-O-TagE (Compound 68)

[0331]

[0332] H-Ala-O-TagE (compound 67) was dissolved in THF-DMF (10 mL, 9:1) and stirred for 15 min with Fmoc-Ala-OH·HO (214.1 mg, 0.65 mmol), DIPEA (280 μL, 1.65 mmol), and COMU (278.4 mg, 0.65 mmol). 1-Methylpiperazine (24.8 μL, 0.23 mmol) was added to the reaction solution and stirred for 10 min. 1-Methylpiperazine (1.1 mL, 10 mmol) and DBU (522 μL, 3.5 mmol) were added and stirred for 10 min. 0.7 M hydrochloric acid (32 mL) was then added, and the mixture was extracted three times with THF-cyclohexane (20 mL, 4:1). The mixture was washed with saturated aqueous sodium bicarbonate (30 mL) and saturated brine (30 mL), and dried over sodium sulfate. The solvent was removed under reduced pressure to give H-Ala-Ala-O-TagE. The above procedure was then repeated using Fmoc-Ala-OH to give 498.8 mg (0.35 mmol, 70% yield) of Fmoc-Ala-Ala-Ala-O-TagE (Compound 68). Exact Mass [M+H] + calculated for [C 85 H 141 N4O 13 ] + 1426.1, observed 1426.5. The HPLC measurement conditions were as follows (FIG. 16): Solvent A: THF, Solvent B: 0.1% TFA MQ water Solvent ratio: A:B = 70:30 → 95:5 (15 min) Column: Nomura Chemical Develosil C8 150 x 2.0 mm, 3 μm Flow rate: 0.20 mL / min Column temperature: 40 o C

[0333] A comparative experiment of the condensation reaction with conventional tags was carried out as follows. The condensation reaction was carried out according to the method described herein, and the HPLC measurement conditions for product detection were as follows: Solvent A: THF, Solvent B: 0.1% TFA MQ water, Solvent ratio: A:B = 75:25, Column: Nomura Chemical ODS-UG-5, inner diameter 2.0 mm, length 150 mm, Flow rate: 0.20 mL / min, Column temperature: 40o C

[0334] Fmoc-Ile-OH was introduced into the tag of the comparative example (hereinafter referred to as the conventional tag) as follows.

[0335]

[0336] 53.0 mg (0.15 mmol) of Fmoc-Ile-OH and 107.0 mg (0.10 mmol) of the conventional tag were dissolved in 2.0 mL of THF-c-Hex (1:1). 2.4 mg (0.020 mmol) of DMAP and 23.1 μL (0.15 mmol) of DIPCI were added and the mixture was stirred for 30 minutes. Since the reaction was not complete, 23.0 μL (0.15 mmol) of DIPCI and 18.6 mg (0.050 mmol) of Fmoc-Ile-OH were added and the mixture was stirred for 30 minutes. 20 mL of hexane was added to the reaction solution, and the organic layer was washed with 20 mL of 1 N HCl, 20 mL of saturated sodium bicarbonate solution, and 20 mL of saturated brine, dried over sodium sulfate, and evaporated under reduced pressure. The residue was subjected to column chromatography (hexane:ethyl acetate=10:1) to obtain 140.5 mg (yield quant.) of Fmoc-Ile-O-TagF (compound 69).

[0337] Synthesis of Fmoc-Ile-O-TagD (Compound 70)

[0338]

[0339] 53.0 mg (0.15 mmol) of Fmoc-Ile-OH and 76.1 mg (0.10 mmol) of compound 29 were dissolved in 2.0 mL of THF-c-Hex (1:1). 2.4 mg (0.020 mmol) of DMAP and 23.1 μL (0.15 mmol) of DIPCI were added and the mixture was stirred for 1 hour. Since the reaction was not complete, 7.7 μL (0.050 mmol) of DIPCI and 18.6 mg (0.05 mmol) of Fmoc-Ile-OH were added and the mixture was stirred for 10 minutes. After confirming completion of the reaction, 4.0 mL each of c-Hex and MeCN were added, and the mixture was separated into two phases. After collecting the c-Hex phase, an additional 4.0 mL of c-Hex was added, and the same extraction procedure was performed. This procedure was repeated three times, and the solvent was removed under reduced pressure. The residue was subjected to column chromatography (hexane:ethyl acetate=7:1) to obtain 105.0 mg (yield 96%) of Fmoc-Ile-O-TagD (compound 70).

[0340] The results are shown in Figure 17. Figure (a) shows the chromatography result of compound 70, where a single peak is observed. Figure (b) shows the chromatography result of compound 69, which has a branched chain, where at least two peaks are observed. These results prove that the compounds of the present invention are highly pure compounds obtained during the synthesis process.

Claims

1. Formula (I): (wherein X is (CH2)m, CH(CH3), or C(CH3)2; Y is a hydroxy group, a thiol group, an amino group, an alkylamino group, a formyl group, or a carboxy group; R may be the same or different and each independently represents a linear saturated hydrocarbon group having 3 to 30 carbon atoms and containing one or more heteroatoms; n is an integer of 5 or less; and m is 0 or a natural number from 1 to 3.) 2. The alkoxy-substituted aromatic compound of claim 1, wherein said heteroatom is oxygen.

3. The alkoxy-substituted aromatic compound of claim 1, wherein the number of heteroatoms is 5 or less.

4. The alkoxy-substituted aromatic compound of claim 1, wherein m is 1.

5. The alkoxy-substituted aromatic compound of claim 2, wherein R is selected from the following formulas:

6. The alkoxy-substituted aromatic compound of claim 3, wherein the number of heteroatoms is 1 or 2.

7. The alkoxy-substituted aromatic compound of claim 1, wherein in said R, one said heteroatom is bonded to at least four -CH2- groups.

8. A separation carrier which is the alkoxy-substituted aromatic compound according to claim 1.

9. A reagent for peptide synthesis comprising the alkoxy-substituted aromatic compound according to any one of claims 1 to 8.

10. An amino acid-added alkoxy-substituted aromatic compound obtained by adding an amino acid to the alkoxy-substituted aromatic compound according to claim 1.

11. A method for isolating a protected amino acid or a protected peptide protected on a carrier from an unsaturated alkyl-containing aromatic compound represented by formula (I), comprising the steps of: (wherein X is (CH2)m, CH(CH3), or C(CH3)2; Y is a hydroxy group, a thiol group, an amino group, an alkylamino group, a formyl group, or a carboxy group; R may be the same or different and each independently represents a straight-chain saturated hydrocarbon group having 3 to 30 carbon atoms and containing one or more heteroatoms; n is an integer of 5 or less, and m is 0 or a natural number of 1 to 3), to obtain an N-protected amino acid whose N-terminus is protected with a carrier ("N-protected") derived from the alkoxy-substituted aromatic compound, or to obtain a C-protected-N-protected peptide by condensing an N-protected amino acid with a C-protected amino acid or a C-protected peptide; step (2): removing the protecting group at the N-terminus of the carrier-protected-N-protected amino acid or carrier-protected-N-protected peptide obtained in step (1); and Step (3): separating the carrier-protected amino acid or carrier-protected peptide obtained in Step (2).

12. The separation method according to claim 10, wherein step (3) is carried out by either (i) a solid-liquid separation step in which the supported amino acid or supported peptide is insolubilized (crystallized) and then separated, or (ii) a liquid separation step in which an organic solvent containing the supported amino acid or supported peptide is separated from an aqueous solution or organic solvent containing water or an organic solvent containing impurities.

13. The separation method according to claim 10, wherein the N-protected amino acid or N-protected peptide is an amino acid or peptide whose amino group is protected with a t-butoxycarbonyl (Boc) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a benzyloxycarbonyl (Cbz) group, a 2-(trimethylsilyl)ethoxycarbonyl (Teoc) group, or the like.

14. The separation method according to claim 10, wherein the organic solvent or mixture of organic solvents is at least one organic solvent selected from the group consisting of THF, DMF, pentane, toluene, normal hexane, heptane, cyclohexane, CPME, MTBE, 2-methyl THF, 4-methyl THP, isopropyl acetate, DCM, chloroform, DMSO, 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, methanol, acetonitrile, ethyl acetate, butyl acetate, and anisole, or a mixture of two or more thereof.

15. A method for synthesizing a peptide in a liquid phase, comprising the following steps: Step (A): In an organic solvent or a mixture of organic solvents, reacting a compound represented by the following formula (I): (wherein X is (CH2)m, CH(CH3), or C(CH3)2; Y is a hydroxy group, a thiol group, an amino group, an alkylamino group, a formyl group, or a carboxy group; R may be the same or different and each independently represents a straight-chain saturated hydrocarbon group having 3 to 30 carbon atoms and containing one or more heteroatoms; n is an integer of 5 or less, and m is a natural number of 0 or 1 to 3), and an amino acid or N-protected peptide in which the amino group at the N-terminus is protected (N-protected) in the presence of a condensing agent to obtain a C-protected-N-protected peptide; step (B): deprotecting the protecting group at the N-terminus of the C-protected-N-protected peptide to obtain a C-protected peptide; and step (C): isolating the C-protected peptide.

16. The peptide synthesis method according to claim 14, wherein the N-protected amino acid or N-protected peptide is an amino acid or peptide whose amino group is protected with a t-butoxycarbonyl (Boc) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a benzyloxycarbonyl (Cbz) group, a 2-(trimethylsilyl)ethoxycarbonyl (Teoc) group, or the like.

17. The peptide synthesis method according to claim 14, wherein the organic solvent or mixture of organic solvents is at least one organic solvent selected from the group consisting of THF, DMF, pentane, toluene, normal hexane, heptane, cyclohexane, CPME, MTBE, 2-methyl THF, 4-methyl THP, isopropyl acetate, DCM, chloroform, DMSO, 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, methanol, acetonitrile, ethyl acetate, butyl acetate, and anisole, or a mixture of two or more thereof.

18. The peptide synthesis method according to claim 14, further comprising the following step (a1) after step (A): adding a water-soluble amine to the reaction solution after the condensation reaction.

19. The peptide synthesis method according to claim 14, wherein step (B) is a step of carrying out deprotection in the presence of a water-soluble amine.

20. The peptide synthesis method according to claim 14, wherein step (C) is a step of neutralizing the reaction solution by adding an acid, washing with an acidic aqueous solution, separating the reaction solution, removing the aqueous layer, obtaining an organic layer, and isolating the C-carrier-protected peptide.

21. The method for synthesizing a peptide according to claim 14, which comprises repeating the step (C) one or more times using the C-carrier protected peptide separated in the step (C).

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