Novel tags for liquid-phase peptide synthesis

Abstract

The present invention relates to the compound described in formula (I), where X is -O-(CH2) n -Selected from the group consisting of (where n is 0 to 4 in the formula), R 1 and R 2 C 11 ~C 25 SP is a spacer selected from the group consisting of alkyls, in particular from the group consisting of saturated C4-C8 alkyls, unsaturated C4-C8 alkyls and substituted or unsubstituted C6-aromatic compounds, in particular from the group consisting of alkyl-substituted C6-aromatic compounds, Y is a heteroatom selected from the group consisting of -O- and -NH-, L is a linker for peptide synthesis, and m is 0 or 1, in particular from the group consisting of 1. [Formula 1] JPEG2026511000000030.jpg2356

Description

[Technical Field]

[0001] This invention relates to a novel tag for liquid-phase peptide synthesis. [Background technology]

[0002] Merrifield's 1963 invention of solid-phase peptide synthesis (SPPS) revolutionized peptide chemistry through the introduction of resins, enabling coupling and deprotection reactions in heterogeneous systems, and thus allowing for the removal of excess reagents and byproducts through simple washing steps (Merrifield, RB, J.Am.Chem.Soc. 1963, 85(14), 2149-2154). While efficient SPPS requires excess reagents and large amounts of solvent, the commonly used Fmoc-tBu strategy can be easily scaled up and automated for large-scale peptide production. However, in an environmentally friendly chemistry context, the large amount of waste generated in SPPS and the use of hazardous solvents such as DMF, NMP, and DCM must be addressed. In contrast, liquid-phase peptide synthesis (LPPS) represents an approach that meets these requirements, as the reaction takes place in solution using a soluble tag attached to the growing peptide chain (see Albericio, F et al., Chem. Rev. 2022, 16, 13516-13546 for a review). LPPS can be carried out using less solvent and excess reagents, while also offering the possibility of introducing environmentally friendly solvents such as tetrahydrofuran (Chiba, K. Tamaki, H et al., Org. Process Res. Dev. 2019, 23, 11, 2576-2581) and cyclopentyl methyl ether (Kubota, H et al., Molecules 2021, 26(12), 3497).Starting from the initial PEG-based approach by Mutter and Bayer (Bayer, E and Mutter, M, Nature 1972, 237, 512 - 513; Mutter, M and Bayer, E, Angew. Chem., Int. Ed. Engl. 1974, 13, 88 - 89), Tamaki et al. (Tamaki, H et al., Bull. Chem. Soc. Jpn. 2001, 74, 733 - 738) invented substituted hydrophobic benzyl alcohol (HAB) for tag-assisted LPPS, which was improved by different groups (e.g., Sunazuka, T et al., Tetrahedron 2011, 67, 6633 - 6643 and Takahashi, D et al., Angew. Chem., Int. Ed. 2017, 56, 7803 - 7807). These studies usually used the Fmoc approach to successfully use soluble HAB tags for the synthesis of small to medium-sized peptides while achieving the purification of the desired peptide by precipitation or membrane-assisted filtration and in some cases aqueous post-treatment procedures.

[0003] Based on the above state-of-the-art technology, the object of the present invention is to provide means and methods for novel tags for peptide synthesis in solution. This object is achieved by the subject matter of the independent claims of this specification, as well as by the further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

Summary of the Invention

[0004] The first aspect of the present invention relates to a compound described by formula (I):

Chemical Formula

[0005] Terms and Definitions For the purposes of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular always include the plural and vice versa. If any of the definitions described below conflict with any document incorporated herein by reference, the defined description shall prevail.

[0006] As used herein, the terms "comprising," "having," "containing," and "including," and other similar forms, as well as their grammatical equivalents, are equivalent in meaning and are intended to be non-limiting in that they do not mean that one or more items following any one of these words are an exhaustive listing of such one or more items or are limited to only the one or more items listed. For example, an article "comprising" components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. Thus, "comprises" and its similar forms, as well as their grammatical equivalents, are intended to and are understood to include disclosure in the form of "consisting essentially of" or "consisting of."

[0007] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, each intervening value between the upper and lower limits of that range, up to one-tenth of the lower limit, and any other stated or intervening values ​​within that stated range, are understood to be included in this disclosure, subject to any specifically excluded limits within the stated range. If the stated range includes one or both limits, the range excluding one or both of those limits is also included in this disclosure.

[0008] References to values ​​or parameters "about" in this specification include (and describe) variations directed toward the value or parameter itself. For example, the statement "about X" includes the statement "X".

[0009] As used herein, including in the appended claims, the singular forms "a," "or," and "the" refer to multiple subjects unless the context explicitly indicates otherwise.

[0010] As used herein, “and / or” should be interpreted as a specific description of each of two specified features or components, with or without the other. Thus, the term “and / or” as used in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B and / or C” is intended to include each of the following embodiments, namely A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, and organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Ausubel et al., Short Protocols in Molecular Biology (2002), 5th Ed., John Wiley & Sons, Inc.) and chemical methods.

[0012] In the context of this specification, the term "linker" refers to a molecule used in Fmoc-solid-phase peptide synthesis (SPPS) that connects the resin on which SPPS is performed with the formed peptide. The linker can be separated from the peptide via a spacer. Once the desired peptide length is reached, the linker is cleaved from the peptide.

[0013] In the context of this specification, the term "spacer" refers to a flexible hydrophobic compound consisting of hydrocarbon atoms that connect a linker molecule to a tag core.

[0014] In the context of this specification, the term "tag" refers to a molecule on which a peptide sequence is formed. Tags are typically attached to the N-terminus or C-terminus of the formed peptide.

[0015] The chemical formulas herein follow the conventions of organic chemistry so as not to show hydrogen atoms on a carbon scaffold. Carbon is tetravalent, and unshown bonds are assumed to be hydrogen unless otherwise specified. Hydrogen can be exchanged for deuterium without altering the bulk chemical properties of the molecule. However, in the case of dye molecules or drug molecules, the exchange of hydrogen for deuterium may result in changes to the molecular's spectral properties or receptor interactions. Unless otherwise expressly stated herein, disclosures of chemical formulas, explicitly or implicitly indicated by the conventions reiterated in the first sentence of this paragraph, include molecules in which one or more hydrogen atoms have been exchanged for deuterium.

[0016] In the context of this specification, the term "saturated alkyl" refers to saturated linear or branched hydrocarbons.

[0017] In the context of this specification, the term "unsaturated alkyl" refers to a straight-chain or branched-chain hydrocarbon in which one or more carbon-carbon bonds may be unsaturated.

[0018] In the context of this specification, the term alkyl refers to saturated linear, branched, or cyclic hydrocarbons consisting of a specified number of carbon atoms. Examples of alkyl residues include, but are not limited to, n-butyl, isobutyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1,2-dimethylpropyl, cyclopentyl, cyclohexyl, methylcyclopentyl, methylcyclohexanyl, heptyl, decanyl, and dodecyl. In certain embodiments, a C5 alkyl is a pentyl or cyclopentyl moiety, a C6 alkyl is a hexyl or cyclohexyl moiety, and a C7 alkyl is a heptyl or cycloheptyl, and this also applies to higher alkyls as commonly understood by those skilled in the art.

[0019] The term "alkyl-substituted C6-aryl" in the context of this specification relates to cyclic aromatic C6 hydrocarbons. The C6-aryl may be further substituted with one or several carbon atoms by a group selected from saturated or unsaturated hydrocarbons (alkyl in its broadest sense). Examples include, but are not limited to, phenyl, benzyl, xylylenyl, and toluenyl.

Embodiments for Carrying Out the Invention

[0020] The first aspect of the present invention relates to a compound described by formula (I):

Chemical formula

[0021] The compound is used as a tag for performing peptide elongation in a more environmentally friendly solvent compared to SPPS. Furthermore, the novel tag enables simple post-treatment of the coupling reaction by simple phase separation. The obtained tag-conjugated peptide remains in the organic phase and can be directly used in the next coupling step without further purification steps. The synthesis of the desired tag is established from readily available and inexpensive starting materials, providing inexpensive molecules for LPPS.

[0022] In certain embodiments, n is 0 or 1.

[0023] In certain embodiments, n is 0.

[0024] In a particular embodiment, R 1 and R 2 These are independent of each other, branching or substituting C 11 ~C 25 It is alkyl.

[0025] In a particular embodiment, R 1 and R 2 Independently, C 13 ~C 25 Selected from the group consisting of alkyl groups.

[0026] In a particular embodiment, R 1 C 17 ~C 25 Selected from the group consisting of alkyl groups.

[0027] In a particular embodiment, R 1 C 21 ~C 25 Selected from the group consisting of alkyl groups.

[0028] In a particular embodiment, R 2 C 17 ~C 25 Selected from the group consisting of alkyl groups.

[0029] In a particular embodiment, R 2 C 21 ~C 25 Selected from the group consisting of alkyl groups.

[0030] In a particular embodiment, R 1 and R 2 This is a branch or substitution C 11 ~C 25 It is alkyl.

[0031] In a particular embodiment, R 1and R 2 C 13 ~C 25 Selected from the group consisting of alkyl groups.

[0032] In a particular embodiment, R 1 and R 2 C 17 ~C 25 Selected from the group consisting of alkyl groups.

[0033] In a particular embodiment, R 1 and R 2 C 21 ~C 25 Selected from the group consisting of alkyl groups.

[0034] In certain embodiments, Y1 is -NH- and Y2 is -NH- or -O.

[0035] In a particular embodiment, Y1 is -NH- and Y2 is -NH-.

[0036] In certain embodiments, SP is selected from the group consisting of saturated C4-C8 alkyl, unsaturated C4-C8 alkyl, and substituted C6-aromatic compounds.

[0037] In certain embodiments, SP is selected from the group consisting of saturated C4-C8 alkyl and alkyl-substituted C6 aromatic compounds.

[0038] In certain embodiments, SP is selected from the group consisting of saturated C4-C8 alkyl groups.

[0039] In certain embodiments, SP is selected from the group consisting of saturated C5-C7 alkyl and alkyl-substituted C6 aromatic compounds.

[0040] In certain embodiments, SP is selected from the group consisting of saturated C5-C7 alkyl groups.

[0041] In certain embodiments, linker L is selected from the group consisting of Rink, Sieber, Trityl, Ramage, Wang, Sasrin, HMPB, 2-chlorotrityl, MBHA, PAL, HMBA, and HMPA.

[0042] In certain embodiments, linker L is selected from the group consisting of Ramage, Rink, HMPA, PAL, and HMBA. In certain embodiments, linker L is selected from the group consisting of Rink, Sieber, Trityl, Ramage, Wang, Sasrin, HMPB, 2-chlorotrityl, and MBHA. In certain embodiments, linker L is selected from the group consisting of Rink, Sieber, and Trityl.

[0043] The tags can be used for peptide synthesis, and compared to conventional solid-phase peptide synthesis, peptide synthesis is performed in a more environmentally friendly solvent and with less solvent, making this approach a more environmentally friendly and cost-effective approach to peptide synthesis.

[0044] Furthermore, the use of tags in peptide synthesis eliminates the need for additional purification steps and results in higher peptide yields compared to conventional peptide synthesis.

[0045] The present invention is further illustrated by the following examples from which further embodiments and advantages can be derived. These examples are illustrative and not intended to limit the scope of the present invention.

[0046] example Example 1: Tag synthesis Preparation of 2-chloro-4,6-bis(heptadecan-9-yloxy)-1,3,5-triazine [ka] Under a nitrogen atmosphere, heptadecane-9-ol (48.68 g, 3.5 equivalents, 189.80 mmol) was dissolved in dried, stabilized tetrahydrofuran (500 mL), and sodium hydride (11.00 g, 60% by weight, 5.0 equivalents, 271.10 mmol) was gradually added at 4°C for 10 minutes. After the addition was complete, the reaction was allowed to return to room temperature and stirred for 1 hour. Then, 2,4,6-trichloro-1,3,5-triazine (10.00 g, 1.0 equivalent, 54.23 mmol) was gradually added over 10 minutes, and the mixture was stirred at 40°C for 40 hours.

[0047] The reaction was stopped for 15 minutes by adding water (100 mL) in an ice bath, and then diluted with ethyl acetate (200 mL). The pH was adjusted to 5 by adding 1 M HCl. The mixture was transferred to a separatory funnel and extracted. The combined organic phase was washed with brine (100 mL) and dried over MgSO4. The solvent was removed under reduced pressure to obtain the crude product (16.24 g, 26.0 mmol, 48.0%) as a white waxy powder.

[0048] The crude product was further purified by column chromatography (1000 g, SiO2; siRNA / n-heptane, 1:50) to obtain a pale yellow oil. MS (ESI, cation): m / z = 624.53 [M + H] +

[0049] Preparation of N1-(4,6-bis(heptadecan-9-yloxy)-1,3,5-triazine-2-yl)hexane-1,6-diamine [ka] Hexane-1,6-diamine (10.24 g, 5.0 equivalents, 88.079 mmol) was dissolved in toluene (200 mL) and stirred at room temperature for 1 hour. 2-Chloro-4,6-bis(heptadecane-9-yloxy)-1,3,5-triazine (11.00 g, 1.0 equivalent, 17.616 mmol) was dissolved in toluene (100 mL) and added to the reaction mixture. Then, diisopropylethylamine (17.00 g, 23 mL, 7.6 equivalents, 0.13 mol) was added, and the reaction mixture was stirred at 80°C for 48 hours. The solvent was removed under reduced pressure. The mixture was diluted in 200 mL of ethyl acetate, transferred to a separatory funnel, and extracted three times with water (100 mL) and brine (50 mL). The solvent was removed under reduced pressure to obtain the crude product (9.85 g, 13.98 mmol, 79.4%) as a yellowish oil. The product was used without further purification. MS (ESI, cation): m / z = 704.66 [M + H] +

[0050] Preparation of (9H-fluoren-9-yl)methyl((4-(2-((6-((4,6-bis(heptadecan-9-yloxy)-1,3,5-triazine-2-yl)amino)hexyl)amino)2-oxoethoxy)phenyl)(2,4-dimethoxyphenyl)methyl)carbamate [ka] Fmoc-Rink-Linker (15.33 g, 2.0 equivalents, 28.40 mmol) and HOBt (4.86 g, 2.0 equivalents, 28.40 mmol) were dissolved in dichloromethane (250 mL), and EDC HCl (5.50 g, 99% by weight, 2.0 equivalents, 28.40 mmol) was added gradually over 5 minutes. After stirring at room temperature for 1 hour, the reaction mixture was cooled in an ice bath (internal temperature 4°C), and 4-methylmorpholine (2.87 g, 3.2 mL, 2.0 equivalents, 28.40 mmol) was added. Subsequently, N 1-(4,6-bis(heptadecan-9-yloxy)-1,3,5-triazine-2-yl)hexane-1,6-diamine (10.00 g, 1.0 equivalent, 14.20 mmol) was added gradually over 5 minutes while maintaining the internal temperature below 10°C. After the addition was complete, the reaction mixture was allowed to return to room temperature.

[0051] After stirring at 40°C for 8 hours, the suspension was evaporated to remove dichloromethane. The crude product was diluted with ethyl acetate and washed three times with water (100 mL) and once with brine (100 mL) to obtain (9H-fluoren-9-yl)methyl((4-(2-((6-((4,6-bis(heptadecane-9-yloxy)-1,3,5-triazine-2-yl)amino)hexyl)amino)-2-oxoethoxy)phenyl)(2,4-dimethoxyphenyl)-methyl)carbamate (12.52 g, 10.21 mmol, 71.9%). MS (ESI, cation): m / z = 1225.83 [M + H] +

number

[0052] Preparation of (9H-fluoren-9-yl)methyl(2-(2-((6-((4,6-bis(heptadecan-9-yloxy)-1,3,5-triazine-2-yl)amino)hexyl)amino)2-oxoethoxy)-10,11-dihydro-5H-dibenzo[a,d][7]anulen-5-yl)-carbamate [ka] Fmoc-Ramage-linker (3.95 g, 1.1 equivalents, 7.81 mmol) and HOBt (1.46 g, 1.2 equivalents, 8.52 mmol) were dissolved in DMF (40 mL), and the reaction mixture was cooled in an ice bath (internal temperature 4 °C). 4-Methylmorpholine (1.46 g, 1.2 equivalents, 8.52 mmol) was added at 4 °C, followed by the addition of EDC HCl (2.06 g, 99% by weight, 1.5 equivalents, 10.7 mmol) in two separate additions. Subsequently, N 1-(4,6-bis(heptadecane-9-yloxy)-1,3,5-triazine-2-yl)hexane-1,6-diamine (5.00 g, 1.0 equivalent, 7.10 mmol) was dissolved in DMF (31 mL) and added gradually over 3 minutes, while maintaining the internal temperature below 10°C. After the addition was complete, the reaction mixture was allowed to return to room temperature.

[0053] After stirring at 22°C for 3 hours, the crude product was diluted with 71 mL of acetonitrile and cooled to 4°C. 71 mL of water was slowly added until a precipitate formed, and another 71 mL of water was added at 22°C. The precipitate was collected by filtration through P3 glass frit. The precipitate was washed with 70 / 30 water / acetonitrile (100 mL) and 90 / 10 water / acetonitrile (100 mL). The precipitate was then dissolved in 200 mL of 2-methyltetrahydrofuran, washed with brine (100 mL), and dried over MgSO4. The crude substance was purified by MPLC (run four times on a 24g SiO2 column) using a gradient from DCM to DCM / siRNA 80:20 to obtain (9H-fluoren-9-yl)methyl-(2-(2-((6-((4,6-bis(heptadecan-9-yloxy)-1,3,5-triazine-2-yl)amino)hexyl)-amino)-2-oxoethoxy)-10,11-dihydro-5H-dibenzo[a,d][7]anulen-5-yl)carbamate (5.39g, 4.52 mmol, 63.7%). MS (ESI, cation): m / z = 1191.87 [M + H] +

number

[0054] Preparation of 2,4-bis((2-butyloctyl)oxy)-6-chloro-1,3,5-triazine [ka] Under a nitrogen atmosphere, 2-butyloctan-1-ol (2.02 g, 2.4 mL, 2.0 equivalents, 10.8 mmol) was dissolved in 2-methyltetrahydrofuran (20 mL) at room temperature. The reaction mixture was cooled to 0°C, and sodium hydride (2.17 g, 60% by weight, 10.0 equivalents, 54.20 mmol) was added gradually. After the addition was complete, the reaction mixture was refluxed for 18 hours. After cooling to -10°C, the active alcohol was added dropwise in three portions to a chilled solution (-10°C) of 2,4,6-trichloro-1,3,5-triazine (1.00 g, 1.0 equivalent, 5.42 mmol) dissolved in 2-methyltetrahydrofuran (10 mL). The reaction mixture was stirred and allowed to return to room temperature for 4 hours. The reaction was then stopped with H2O, transferred to a separatory funnel, and washed with brine (50 mL). The organic phase was dried over MgSO4 to obtain 2,4-bis((2-butyloctyl)oxy)-6-chloro-1,3,5-triazine. The product was used without further purification. MS (ESI, cation): m / z = 484.33 [M + H] +

[0055] N 1 Preparation of -(4,6-bis((2-butyloctyl)oxy)-1,3,5-triazin-2-yl)hexane-1,6-diamine [ka] Hexane-1,6-diamine (6.31 g, 10.0 equivalents, 54.30 mmol) was dissolved in 2-methyltetrahydrofuran (30 mL) and stirred at high temperature for 10 minutes. The diamine solution was heated to 70°C and a solution of 2,4-bis((2-butyloctyl)oxy)-6-chloro-1,3,5-triazine (2.63 g, 1.0 equivalent, 5.43 mmol) in 2-methyltetrahydrofuran (20 mL) was added dropwise. After the addition was complete, the reaction mixture was stirred for approximately 1.5 hours. The reaction mixture was then cooled to room temperature and the precipitate was filtered (G3-frit). The remaining solution was diluted with ethyl acetate (600 mL). The organic layer was washed four times with water (4 × 250 mL), once with 0.5 M HCl (250 mL), and once with brine (250 mL). The combined organic phase was dried over MgSO4 and purified by column chromatography (100g, SiO2; DCM:NH3(7N)90:10 in MeOH) to obtain N1-(4,6-bis((2-butyloctyl)oxy)-1,3,5-triazine-2-yl)hexane-1,6-diamine (1.10g, 1.96 mmol, 36.0% in two steps). MS (ESI, cation): m / z = 564.52 [M + H] +

[0056] Preparation of (9H-fluoren-9-yl)methyl((4-(2-((6-((4,6-bis((2-butyloctyl)oxy)-1,3,5-triazine-2-yl)amino)hexyl)amino)-2-oxoethoxy)phenyl)(2,4-dimethoxyphenyl)methyl)carbamate [ka] To a solution of 2-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)(2,4-dimethoxyphenyl)methyl)phenoxy)acetic acid (526 mg, 1.1 equivalents, 975 μmol) in DMF (5 mL), HOBt dihydrate (182 mg, 1.2 equivalents, 1.06 mmol) was added, and the mixture was cooled to 4°C. EDC HCl (258 mg, 99% by weight, 1.5 equivalents, 1.33 mmol), followed by 4-methylmorpholine (359 mg, 0.39 mL, 4.0 equivalents, 3.55 mmol), was added, and the mixture was stirred at 4°C for 5 minutes. Then, N 1 A solution of -(4,6-bis((2-butyloctyl)oxy)-1,3,5-triazine-2-yl)hexane-1,6-diamine (526 mg, 1.1 equivalents, 975 μmol) in DMF (5 mL) was added. The mixture was stirred for a further 5 minutes at 4°C, then heated and stirred at room temperature for 1 hour. The mixture was diluted with acetonitrile (10 mL) and added dropwise to 40 mL of distilled water at 4°C. The resulting mixture was diluted with dichloromethane (150 mL) and 5 wt% LiCl (50 mL), and the mixture was transferred to a separatory funnel. The resulting emulsion could be clarified by adding 20 mL of ethanol. The phases were separated, and the aqueous phase was extracted twice with dichloromethane (50 mL). The combined organic phases were washed with 5 wt% LiCl and brine, then dried over MgSO4, and purified by MPLC (12 g SiO2 column) using a gradient from DCM to DCM / SiO2 80 / 20 to obtain (9H-fluoren-9-yl)methyl((4-(2-((6-((4,6-bis((2-butyloctyl)oxy)-1,3,5-triazine-2-yl)amino)hexyl)amino)-2-oxoethoxy)phenyl)(2,4-dimethoxyphenyl)methyl)carbamate (480 mg, 442 μmol, 49.9%). MS (ESI, cation): m / z = 1085.71 [M + H] +

number

[0057] Preparation of N-(3-((12-azanail)methyl)benzyl)-4,6-bis-(heptadecane-9-yloxy)-1,3,5-triazine-2-amine [ka] 1,3-Phenylenedimethanamine (7.39 g, 7.16 mL, 10.0 equivalents, 54.30 mmol) was dissolved in 2-methyltetrahydrofuran (30 mL) and stirred at high temperature for 10 minutes. A solution of 2-chloro-4,6-bis(heptadecane-9-yloxy)-1,3,5-triazine (3.39 g, 1.0 equivalent, 5.43 mmol) in 2-methyltetrahydrofuran (35 mL) was added dropwise. After the addition was complete, the reaction mixture was stirred for about 1 hour, then cooled to room temperature, and the precipitate was filtered (G3-frit). The remaining solution was diluted with ethyl acetate (600 mL). The organic layer was washed four times with water (4 × 250 mL), once with 0.5 M HCl (250 mL), and once with brine (250 mL), dried over MgSO4, and evaporated. The crude product was further purified by column chromatography (100 g, SiO2;DCM / MeOH, 95:5~91:10, followed by NH3(7N)90:10 in DCM:MeOH) to obtain the product as a pale yellow oil (1.00 g, 1.39 mmol, 25.5%). MS (ESI, cation): m / z = 724.67 [M + H] +

[0058] Preparation of 4-(((triisopropylsilyl)oxy)methyl)benzoic acid [ka] To a suspension of 4-(hydroxymethyl)benzoic acid (0.85 g, 1.0 equivalent, 5.60 mmol) in dichloromethane (30 mL), imidazole (1.10 g, 3.0 equivalent, 17.00 mmol) was added, followed by the dropwise addition of triisopropylsilyl triflate (3.50 g, 3.10 mL, 2.0 equivalent, 11.00 mmol) at 4°C. The mixture was allowed to cool to room temperature and then heated under reflux for 3 hours. After this, the solvent was removed under reduced pressure, and the residue was dissolved in THF / H2O (1:1; 30 mL). Potassium carbonate (0.77 g, 1.0 equivalent, 5.60 mmol) was then added, and the mixture was heated to approximately 50°C for 1 hour. The reaction mixture was transferred to a separatory funnel and acidified to approximately pH 3.5 by adding 5% by weight citric acid. Ethyl acetate was added to separate the phases. The aqueous phase was extracted with ethyl acetate, and the combined organic extract was washed twice with H2O (2 × 30 mL) and once with brine (30 mL). The crude mixture was dried over MgSO4, and the solvent was removed under reduced pressure. Analytically pure samples were obtained by column chromatography, but the bulk material was used in the next step without further purification.

number

[0059] Preparation of N-(3-(((4,6-bis(heptadecan-9-yloxy)-1,3,5-triazine-2-yl)amino)methyl)benzyl)-4-(((triisopropylsilyl)oxy)methyl)benzamide [ka] To a solution of N-(3-(aminomethyl)benzyl)-4,6-bis(heptadecane-9-yloxy)-1,3,5-triazine-2-amine (181 mg, 1.0 equivalent, 250 μmol) in dichloromethane (10 mL), 4-(((triisopropylsilyl)oxy)methyl)-benzoic acid (92.5 mg, 1.2 equivalents, 300 μmol) and 1H-benzo[d][1,2,3]triazole-1-ol hydrate (49.8 mg, 1.3 equivalents, 325 μmol) were added, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (71.9 mg, 1.50 equivalents, 375 μmol) and 4-methylmorpholine (0.13 g, 0.14 mL, 5.1 equivalents, 1.30 mmol). The mixture was stirred at room temperature for 2.5 hours. The reaction mixture was then transferred to a separatory funnel and washed with 1 M KHSO4, saturated NaHCO3, and brine. The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure. The crude product was further purified by MPLC (4 g SiO2 column) using a gradient from DCM to DCM / siRNA80 / 20 to obtain the product as a pale yellow oil (66 mg, 250 μmol, 26%). MS (ESI, cation): m / z = 10¹⁴.77 [M + H] +

number

[0060] Preparation of 2-chloro-4,6-bis((16-methylheptadecyl)oxy)-1,3,5-triazine [ka] Under a nitrogen atmosphere, 16-methyl-1-heptadecanol (2.93 g, 2.0 equivalents, 10.8 mmol) was dissolved in 2-MeTHF (20 mL), cooled to 0°C, and sodium hydride (2.17 g, 60% by weight, 10.0 equivalents, 54.2 mmol) was added gradually. After the addition was complete, the reaction mixture was refluxed for 18 hours. After cooling to -10°C, the active alcohol was added dropwise over 15 minutes to a chilled solution (-10°C) of 2,4,6-trichloro-1,3,5-triazine (1.00 g, 1.0 equivalent, 5.42 mmol) dissolved in 2-MeTHF (10 mL). The reaction mixture was stirred and allowed to return to room temperature for 1 hour. The reaction was then stopped using cold water, transferred to a separatory funnel, and washed once with brine (50 mL). The organic phase was dried with MgSO4 to obtain 2-chloro-4,6-bis((16-methylheptadecyl)oxy)-1,3,5-triazine. MS (ESI, cation): m / z = 652.62 [M + H] +

[0061] N-(4-(λ 2 Preparation of azanail (butyl)-4,6-bis((16-methylheptadecyl)oxy)-1,3,5-triazine-2-amine [ka] Butane-1,4-diamine (4.78 g, 10.0 equivalents, 54.3 mmol) was dissolved in 2-MeTHF (30 mL) and stirred at high temperature for 10 minutes. 40 mL of 2-MeTHF solution of 2-chloro-4,6-bis((16-methylheptadecyl)oxy)-1,3,5-triazine (3.54 g, 1.0 equivalent, 5.43 mmol) was added dropwise. After complete addition, the reaction mixture was stirred for a further 1.5 hours. The reaction mixture was then cooled to room temperature, and the precipitate was filtered (G3-frit). The remaining solution was diluted with 600 mL of EE. The organic phase was washed with water (4 × 250 mL) and 0.5 M HCl (250 mL). The organic phase was washed with brine (250 mL). The combined organic phase was dried over MgSO4 and evaporated. The crude product was further purified by MPLC (24 g SiO2 column) using a gradient with DCM / MeOH to obtain the product as colorless wax (1.35 g, 1.92 mmol, 35.3% in two steps). MS (ESI, cation): m / z = 704.69 [M + H] +

[0062] Preparation of (9H-fluoren-9-yl)methyl(4-((5-((4-((4,6-bis((16-methylheptadecyl)oxy)-1,3,5-triazine-2-yl)amino)butyl)amino)-5-oxopentyl)oxy)-2,6-dimethoxybenzyl)carbamate [ka] Fmoc-PAL (359 mg, 1.0 equivalent, 710 μmol) was dissolved in DMF (4 mL). Then, HOBt dihydrate (146 mg, 1.2 equivalents, 852 μmol) was added, and the mixture was cooled to 4°C. 4-Methylmorpholine (287 mg, 0.32 mL, 4.0 equivalents, 2.84 mmol) was added at 4°C, followed by the addition of EDC HCl (206 mg, 99% by weight, 1.5 equivalents, 1.07 mmol) in two separate additions. N-(4-(λ 2Azanail)butyl)-4,6-bis((16-methylheptadecyl)oxy)-1,3,5-triazine-2-amine (500 mg, 1.0 equivalent, 710 μmol) was dissolved in DMF (3 mL) and added to the mixture. The reaction mixture was stirred at 4°C for a further 5 minutes, then warmed to room temperature and stirred at this temperature for 1 hour. After a total of 3 hours of stirring, the mixture was diluted with acetonitrile (8 mL) and cooled to 4°C. 20 mL of distilled water was slowly added at 4°C, and an oily precipitate was formed. The precipitate was collected by filtration through P3 glass frit, washed with 100 mL of H2O, dissolved in dichloromethane (100 mL), and washed with brine (100 mL). The organic phase was dried over MgSO4 and the solvent was removed under reduced pressure. The crude product was further purified by MPLC (12 g SiO2 column) using a gradient from DCM to DCM / Â80 / 20 to obtain the product as a pale yellow oil (702 mg, 589 μmol, 83.0%). MS (ESI, cation): m / z = 1191.94 [M + H] +

number

[0063] Preparation of 2-chloro-4,6-bis((2-decyltetradecyl)oxy)-1,3,5-triazine [ka] Under a nitrogen atmosphere, 2-decyltetradecanol (3.85 g, 4.57 mL, 2.0 equivalents, 10.80 mmol) was dissolved in 2-methyltetrahydrofuran (20 mL), cooled to 0°C, and sodium hydride (2.17 g, 60 wt%, 10.0 equivalents, 54.20 mmol) was added little by little. After the addition was complete, the reaction mixture was refluxed for 18 hours. After cooling to -10°C, the active alcohol was added dropwise to a chilled solution (-10°C) of 2,4,6-trichloro-1,3,5-triazine (1.00 g, 1.0 equivalent, 5.42 mmol) dissolved in 2-methyltetrahydrofuran (10 mL). The reaction mixture was stirred and allowed to return to room temperature for 1 hour, then the reaction was stopped with H2O and washed with brine (40 mL). The organic phase was dried with MgSO4, and the solvent was removed under vacuum to obtain 2-chloro-4,6-bis((2-decyltetradecyl)oxy)-1,3,5-triazine. MS (ESI, cation): m / z = 820.75 [M + H] +

[0064] N 1 Preparation of (4,6-bis((2-decyltetradecyl)oxy)-1,3,5-triazine-2-yl)octane-1,8-diamine [ka] Octane-1,8-diamine (7.82 g, 10.0 equivalents, 54.2 mmol) was dissolved in 2-methyltetrahydrofuran (30 mL) and stirred at high temperature for 10 minutes. The diamine solution was heated to 70°C, and a solution of 2-chloro-4,6-bis((2-decyltetradecyl)oxy)-1,3,5-triazine (4.45 g, 1.0 equivalent, 5.42 mmol) in 2-methyltetrahydrofuran (40 mL) was added dropwise. The reaction mixture was stirred for approximately 1.5 hours, then cooled to room temperature, and the precipitate was filtered (G3-frit). The remaining solution was diluted with ethyl acetate (300 mL). The organic phase was washed four times with water (4 × 250 mL), once with 0.5 M HCl (250 mL), and once with brine (250 mL). The combined organic phase was dried over MgSO4 and evaporated. The crude product was further purified by MPLC (24g SiO2 column) using a gradient with DCM / MeOH, and then N 1 -(4,6-bis((2-decyltetradecyl)oxy)-1,3,5-triazine-2-yl)octane-1,8-diamine was obtained as a colorless wax (650 mg, 700 μmol, 12.9% in two steps). MS (ESI, cation): m / z = 928.88 [M + H] +

[0065] Preparation of 2-(4-(((triisopropylsilyl)oxy)methyl)phenoxy)acetic acid (HMPA-TIPS) [ka] To a suspension of 2-(4-(hydroxymethyl)phenoxy)acetic acid (0.50 g, 1.0 equivalent, 2.70 mmol) in dichloromethane (20 mL), imidazole (0.56 g, 3.0 equivalent, 8.20 mmol) was added, followed by the dropwise addition of triisopropylsilyl triflate (2.10 g, 1.80 mL, 2.5 equivalent, 6.90 mmol) at 4°C. The mixture was allowed to return to room temperature and then stirred for 3 days. After this, the suspension was heated under reflux for 2 hours, and the solvent was removed under reduced pressure. The residue was dissolved in THF / H2O (1:1; 20 mL). Potassium carbonate (0.38 g, 1.0 equivalent, 2.70 mmol) was then added, and the mixture was heated to approximately 50°C for 1 hour. The reaction mixture was transferred to a separatory funnel and acidified to approximately pH 3.5 by adding 5% by weight citric acid. Ethyl acetate (15 mL) was added and the phases were separated. The aqueous phase was extracted with ethyl acetate, and the combined organic phase was washed twice with H2O (2 × 20 mL) and once with brine (20 mL). The crude mixture was dried over MgSO4, and the solvent was removed under reduced pressure to obtain 2-(4-(((triisopropylsilyl)oxy)methyl)phenoxy)acetic acid, which was used without further purification.

number

[0066] Preparation of N-(8-((4,6-bis((2-decyltetradecyl)oxy)-1,3,5-triazine-2-yl)amino)octyl)-2-(4-(((triisopropylsilyl)oxy)methyl)phenoxy)acetamide [ka] HMPA-TIPS (492 mg, 3.0 equivalents, 1.45 mmol) was dissolved in dichloromethane (8 mL), and HOBt dihydrate (99.5 mg, 1.2 equivalents, 582 μmol) was added. The mixture was cooled to 4°C. 4-Methylmorpholine (196 mg, 0.22 mL, 4.0 equivalents, 1.94 mmol) was added at 4°C, followed by the addition of EDC HCl (141 mg, 99% by weight, 1.5 equivalents, 727 μmol) in two separate additions. 1-(4,6-bis((2-decyltetradecyl)oxy)-1,3,5-triazine-2-yl)octane-1,8-diamine (450 mg, 1.0 equivalent, 485 μmol) was dissolved in dichloromethane (4 mL) and added to the mixture over 3 minutes at 4°C. The mixture was stirred for a further 5 minutes, then heated to room temperature and stirred at this temperature for 1.5 hours. The mixture was diluted with acetonitrile (3 mL) and distilled water (20 mL) was added. The resulting two-phase mixture was transferred to a separation funnel. 100 mL of brine was added and the aqueous phase was separated. The organic phase was dried over MgSO4 and the solvent was removed under reduced pressure. The crude product was further purified by MPLC (12 g SiO2 column) using a gradient with DCM / EE to obtain the product as a pale yellow oil (217 mg, 174 μmol, 35.8%). MS (ESI, cation): m / z = 1249.15 [M + H] +

number

[0067] Example 2: Peptide synthesis in solution Deprotection Fmoc protective tags (1 equivalent) were dissolved in ethyl acetate (1%). Then, diethylamine (4 equivalents) was added and the mixture was stirred overnight.

[0068] One equivalent of a TIPS protection tag was dissolved in 1% THF. Then, two equivalents of TBAF were added, and the mixture was stirred for 2 hours.

[0069] Coupling The deprotected tag was dissolved in ethyl acetate (1%). Then, Fmoc-protected amino acid (Fmoc-AA-OH) (2 equivalents) and Oxyma (2 equivalents) were added and the mixture was stirred for 1 minute. Next, N,N'-diisopropylcarbodiimide (3 equivalents) was added and the mixture was stirred overnight.

[0070] extraction The reaction mixture was transferred to a separatory funnel and washed with water (10 volume equivalents) and brine (10 volume equivalents). The tags remained in the organic phase and could be used in the subsequent coupling step in solution.

Claims

1. The compound described in formula (I), 【Chemistry 1】 During the ceremony, X is -O-(CH ２ ) ｎ - Selected from the group consisting of (where n is between 0 and 4 in the formula), R １ and R ２ C is independent １１ ~C ２５ Selected from the group consisting of alkyl groups, The SP is, in particular, a saturated C ４ ~C ８ alkyl, an unsaturated C ４ ~C ８ alkyl and a substituted or unsubstituted C ６ -aromatic compound, and in particular, the substituted C ６ -aromatic compound is an alkyl-substituted C ６ -aryl, Y is a heteroatom selected from the group consisting of -O- and -NH- in particular. L is a linker for peptide synthesis, m is 0 or 1, and in particular m is 1. compound.

2. The compound according to claim 1, wherein n is 0.

3. R １ and R ２ These are independent of each other, branching or substituting C １１ ~C ２５ The compound according to claim 1 or 2, wherein it is alkyl.

4. R １ and R ２ C is independent １３ ~C ２５ A compound according to any one of claims 1 to 3, selected from the group consisting of alkyl groups.

5. R １ is C １７ ~C ２５ A compound according to any one of claims 1 to 4, selected from the group consisting of alkyl groups.

6. R ２ is C １７ ~C ２５ A compound according to any one of claims 1 to 5, selected from the group consisting of alkyl groups.

7. Y １ is -NH-, Y ２ The compound according to any one of claims 1 to 6, wherein is -NH- or -O.

8. Y １ is -NH-, Y ２ The compound according to any one of claims 1 to 7, wherein is -NH-.

9. SP is saturated C ４ ~C ８ Alkyl, unsaturated C ４ ~C ８ Alkyl and substituted C ６ - A compound selected from the group consisting of aromatic compounds, according to any one of claims 1 to 8.

10. SP is saturated C ４ ~C ８ Alkyl and substituted C ６ - A compound selected from the group consisting of aromatic compounds, according to any one of claims 1 to 9.

11. SP is C ４ ~C ８ A compound according to any one of claims 1 to 10, selected from the group consisting of alkyl groups.

12. The linker L is selected from the group consisting of Rink, Sieber, Trityl, Ramage, Wang, Sasrin, HMPB, 2-chlorotrityl, MBHA, PAL, HMBA, and HMPA, according to any one of claims 1 to 11.

13. The linker L is selected from the group consisting of Ramage, Rink, HMPA, PAL, and HMBA, as described in any one of claims 1 to 12.

14. The linker L is selected from the group consisting of Rink, Sieber, Trityl, Ramage, Wang, Sasrin, HMPB, 2-chlorotrityl, and MBHA, as described in any one of claims 1 to 11.

15. The compound according to claim 14, wherein the linker L is selected from the group consisting of Rink, Sieber, and Trityl.

Images