Linker molecule for preparing cell membrane-permeable cyclic peptides and use thereof

Abstract

Provided is a linker molecule for producing a cell membrane-permeable ring-shaped peptide, the linker molecule having a cell membrane permeability-imparting group; and a ring-forming group which forms a peptide into a ring shape.

Description

[Technical field]

[0001] The present invention relates to a linker molecule for producing a cell membrane-permeable cyclic peptide, a peptide complex into which the linker molecule has been introduced and a method for producing the same, a peptide library containing the peptide complex, and a method for screening functional peptides using the peptide library. [Background technology]

[0002] In recent years, it has become common to select therapeutic drugs for specific diseases or molecules with high affinity for target molecules from peptide (polypeptide) libraries with various amino acid sequences.

[0003] As a method for producing the peptide library, for example, a ribosome display method (RD method) using a ribosome display complex containing a peptide chain, an mRNA molecule, and a ribosome is known as an in vitro translation system (see, for example, Patent Document 1). In the RD method, as long as there is an in vitro translation system and mRNA, the peptide library can be produced in 10 12 This is an extremely useful tool that can generate peptide libraries of over 100 types in just a few minutes.

[0004] On the other hand, attention is also being paid to the development of medicines in which polypeptides such as antibodies are chemically modified with functional molecules to expand their functions. For example, it is known that peptides can be cyclized to improve their stability in the body, and that cyclization can stabilize their structure, improving their affinity and selectivity for target compounds, and sometimes even resulting in resistance to degradative enzymes and cell membrane permeability.

[0005] In addition, in the development of pharmaceuticals, it is also important to be able to efficiently take up various active ingredients such as proteins and nucleic acids into cells.

[0006] Known techniques for introducing active ingredients into cells include, for example, a method of fusing the amino acid sequence of a cell membrane-permeable peptide that contains a large amount of basic amino acids, and a method of using a dendrimer, a dendritic polymer having a structure that branches regularly from the center (see, for example, Patent Document 2).

[0007] Therefore, molecules that have been given cell membrane permeability by conferring peptides with a cyclic structure are thought to be valuable drug discovery seeds, and peptide libraries containing such peptides will be extremely useful tools in their exploration.

[0008] It has been known that a technique for simply and easily cyclizing (modifying) the peptide chain in the ribosome complex involves modification with a modifying reagent at a specific time, which allows the adhesive function of the ribosome that binds RNA to the peptide translated from it to be maintained without losing its original function (see, for example, Patent Document 3).

[0009] Furthermore, a technique for cyclizing a peptide and imparting cell membrane permeability in a stepwise manner has been reported (see, for example, Non-Patent Document 1).

[0010] However, a technology that can simultaneously cyclize a peptide and confer cell membrane permeability has not yet been developed, and there is currently a strong demand for its prompt development. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] JP 2008-271903 A [Patent Document 2] U.S. Patent No. 7,862,807 [Patent Document 3] International Publication No. 2017 / 213158 [Non-patent literature]

[0012] [Non-Patent Document 1] Dr. Naila Assem, David J. Ferreira, Prof.Dr. Dennis W. Wolan, Prof.Dr. Philip E. Dawson, Acetone-Linked Peptides: A Convergent Approach for Peptide Macrocyclization and Labeling, Angewandte Chemie International Edition, 2015, 54, 8665-8668 Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention aims to solve the above-mentioned problems in the prior art and to achieve the following objects: That is, the present invention aims to provide a linker molecule for preparing a cell membrane-permeable cyclic peptide that can simultaneously cyclize a peptide and impart cell membrane permeability to the peptide, a peptide complex into which the linker molecule has been introduced and a method for producing the same, a peptide library containing the peptide complex, and a method for screening functional peptides using the peptide library. [Means for solving the problem]

[0014] Means for Solving the Problems The present inventors conducted extensive research to solve the above problems, and unexpectedly found that it is possible to synthesize a molecule in which a cell membrane permeability-imparting group and a cyclization group that cyclizes a peptide coexist in the same molecule, and discovered that by using this molecule, it is possible to simultaneously cyclize a peptide and impart cell membrane permeability to the peptide.

[0015] The present invention is based on the findings of the present inventors, and the means for solving the above problems are as follows. <1> The linker molecule for producing a cell membrane-permeable cyclic peptide is characterized by having a cell membrane-permeability imparting group and a cyclization group that cyclizes a peptide. <2> A peptide and the above-mentioned compound introduced into the peptide. <1> and a linker molecule according to any one of claims 1 to 5. <3> The above <2> The peptide library is characterized by comprising the peptide complex according to claim 1. <4> The peptide <1> The present invention relates to a method for producing a peptide conjugate, the method comprising the step of introducing a linker molecule according to the above-mentioned item 1 into the peptide conjugate. <5> The above <3> The present invention relates to a method for screening functional peptides, the method comprising the step of screening for functional peptides using the peptide library described in the above. Effect of the Invention

[0016] According to the present invention, it is possible to solve the above-mentioned problems in the conventional art and achieve the above-mentioned object, and it is possible to provide a linker molecule for producing a cell membrane-permeable cyclic peptide that can simultaneously cyclize a peptide and impart cell membrane permeability to the peptide, a peptide complex into which the linker molecule has been introduced and a method for producing the same, a peptide library containing the peptide complex, and a method for screening functional peptides using the peptide library. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows the results of preparative HPLC of peptide P in Example 2. [Diagram 2] FIG. 2 shows the results of preparative HPLC of the cyclic peptide (peptide complex G3-DCX-P) in Example 2. [Diagram 3] FIG. 3 is a schematic diagram showing the structure of the template DNA used to prepare the ribosome display complex in Example 3. [Figure 4] FIG. 4 shows the results of MALDI-TOF MS measurement in Example 4. [Diagram 5] FIG. 5 shows the results of MALDI-TOF MS measurement in Example 5. [Figure 6]FIG. 6 shows the results of MALDI-TOF MS measurement in Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] (Linker molecules for producing cell membrane-permeable cyclic peptides) The linker molecule for producing a cell membrane-permeable cyclic peptide of the present invention (hereinafter, sometimes referred to as a "linker molecule") has at least a cell membrane-permeability imparting group and a cyclization group that cyclizes the peptide, and further has other components such as a linking group as necessary.

[0019] The linker molecule can be represented by the following general formula (1): ABC... General formula (1) In the general formula (1), "A" represents a cyclizing group, "B" represents a linking group or a single bond, and "C" represents a group that imparts cell membrane permeability.

[0020] <Cyclicifying group> The cyclizing group contributes to cyclization of the peptide by reacting with a reactive amino acid residue in the peptide described below. The reactive amino acid residue is an amino acid residue that reacts with the cyclizing group, and may be an amino acid residue that reacts directly with the cyclizing group, or may be an amino acid residue that has been modified so that it can react with the cyclizing group. The cyclizing group is not particularly limited and can be appropriately selected depending on the purpose, but is preferably an electron-withdrawing group.

[0021] -Electron-withdrawing group- The electron-withdrawing group is not particularly limited and can be appropriately selected depending on the purpose, but preferably contains a halogen.

[0022] The type of the halogen is not particularly limited and can be appropriately selected depending on the purpose.

[0023] The number of the halogens is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 2 or more.

[0024] The electron-withdrawing group preferably has two or more chlorine atoms, more preferably benzyl chloride which may have a substituent, and particularly preferably 3,5-bis(chloromethyl)benzyl group.

[0025] The cyclization of the peptide with the cyclizing group is preferably carried out by reacting the cyclizing group with at least one group selected from the group consisting of a thiol group, an amino group, and a hydroxyl group contained in the peptide.

[0026] <Cell membrane permeabilization group> The cell membrane permeability imparting group is a group that imparts cell membrane permeability to the peptide. The cell membrane permeability imparting group preferably has a basic functional group.

[0027] The form of the cell membrane permeability imparting group is not particularly limited and can be appropriately selected depending on the purpose, but an embodiment having a dendritic structure is preferable. The number of dendritic structural units in the cell membrane permeability imparting group is not particularly limited and may be appropriately selected depending on the purpose, and may be one, or two or more. The number of branches per one dendritic structural unit is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 3 or more. The structure of the branches of the dendritic structure is not particularly limited and can be appropriately selected depending on the purpose.

[0028] -Basic functional group- The basic functional group is not particularly limited and can be appropriately selected according to the purpose, and examples thereof include a guanidino group, an amino group, and an imidazole group. Among these, it is preferable to have a guanidino group. The basic functional group may be used alone or in combination of two or more kinds. The number of the basic functional groups is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 2 or more. The basic functional group preferably has two or more guanidino groups. The position of the basic functional group in the linker molecule is not particularly limited and may be appropriately selected depending on the purpose, but it is preferably located at the end of a branch of the dendritic structure.

[0029] Specific examples of the cell membrane permeability imparting group having a dendritic structure include those represented by the following general formula (C). [ka] The general formula (C) represents a cell membrane permeability-imparting group having a dendritic structure with three branches, and in the formula, "Y" represents a basic functional group. The basic functional groups may be formed at the ends of all the branches, or may be formed at the ends of only a portion of the branches. The cell membrane permeability imparting group may have a plurality of dendritic structures represented by general formula (C), in which case the number of branches may be, for example, 6, 9, or the like.

[0030] <Other configurations> Other configurations in the linker molecule are not particularly limited as long as they do not impair the effects of the present invention and can be appropriately selected depending on the purpose. For example, a linking group and the like can be mentioned.

[0031] <<Connecting group>> The linking group is a group that links the cell membrane permeability imparting group and the cyclizing group. The structure of the linking group is not particularly limited and can be appropriately selected depending on the purpose.

[0032] Specific examples of the linker molecule include compounds represented by the following structural formula: The linker molecule represented by the following structural formula is an example having a 3,5-bis(chloromethyl)benzyl group as a cyclizing group, and having three guanidino groups at the end of a dendritic structure as cell membrane permeability-imparting groups, and the cyclizing group and the cell membrane permeability-imparting groups are linked via a linking group. [ka]

[0033] <Method of producing linker molecule> The method for producing the linker molecule is not particularly limited and can be appropriately selected depending on the purpose. For example, there can be mentioned a method including a cell membrane permeability imparting group precursor forming step, a linking group introducing step of introducing a linking group into the cell membrane permeability imparting group precursor, a step of introducing a cyclizing group precursor into the linking group, and a step of forming a cell membrane permeability imparting group and a cyclizing group from the cell membrane permeability imparting group precursor and the cyclizing group precursor, respectively. Each of the above steps can be carried out by appropriately selecting a known chemical synthesis technique. For example, the cell membrane permeability imparting group precursor forming step can be carried out by the method described in US Pat. No. 7,862,807.

[0034] The method for confirming whether the obtained linker molecule has a desired structure is not particularly limited, and a known analytical method can be appropriately selected, such as mass spectrometry, proton nuclear magnetic resonance spectroscopy, carbon-13 nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, and infrared spectroscopy.

[0035] According to the linker molecule of the present invention, peptide cyclization and imparting cell membrane permeability to the peptide can be carried out in a single step, and therefore these can be achieved easily in a short period of time.

[0036] (Peptide complex) The peptide conjugate of the present invention contains at least a peptide and a linker molecule of the present invention introduced into the peptide, and further contains other components as necessary.

[0037] <Peptide> The peptide is not particularly limited as long as it can be cyclized by introducing the linker molecule, and can be appropriately selected depending on the purpose. The amino acid residue used for introducing the linker molecule (hereinafter, sometimes referred to as "reactive amino acid residue") is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include cysteine ​​residues, lysine residues, serine residues, threonine residues, etc. The reactive amino acid residues may be used alone or in combination of two or more kinds. The number of the reactive amino acid residues in a peptide is preferably 2 or more in terms of cyclizing the peptide. The upper limit of the number of the reactive amino acid residues in a peptide is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 10 or less, since an increase in the number of reactive sites may cause the number and positions of the linker molecules bound to the peptide to become unstable, making it difficult to compare the properties of peptides derived from amino acid sequences. For example, when a cysteine ​​residue in a peptide is involved in stabilizing the higher-order structure of the peptide through a disulfide bond, it is preferable to separately introduce the above-mentioned reactive amino acid residue into the peptide.

[0038] The position of the reactive amino acid residue in the peptide is not particularly limited and can be appropriately selected depending on the purpose.

[0039] For example, when a ribosome display complex (hereinafter sometimes referred to as an "RD complex") containing an mRNA molecule, a peptide chain (hereinafter sometimes referred to as a "polypeptide chain") that is a translation thereof, and a ribosome is used as the peptide, the peptide is preferably located, for example, in the portion protruding from the exit tunnel of the ribosome, specifically between the second position from the N-terminus and the 30th position from the C-terminus (including the second position from the N-terminus and the 30th position from the C-terminus), since the modification reaction by the linker molecule is less likely to be sterically inhibited by the ribosome. The position from the C-terminus is preferably the 50th position, more preferably the 100th position from the C-terminus. Furthermore, when the position of the reactive amino acid residue is counted from the N-terminus, the position can be appropriately set depending on the chain length of the peptide, and is, for example, the 2nd to 1,000th position from the N-terminus, preferably the 2nd to 100th position from the N-terminus, and more preferably the 2nd to 50th position from the N-terminus.

[0040] The method for producing the RD complex is not particularly limited, and a known method can be appropriately selected, for example, the method described in International Publication No. 2017 / 213158. In addition, the RD complex can also be produced using a commercially available kit.

[0041] The amino acid sequence of the peptide is not particularly limited and can be appropriately selected depending on the purpose, but preferably contains a random sequence at a specific position so as to be useful as a peptide library. From such random sequences, a useful amino acid sequence can be identified depending on a predetermined purpose.

[0042] The position of the random sequence in the peptide is not particularly limited and can be appropriately selected depending on the purpose. For example, similar to the position of the reactive amino acid residue, when an RD complex is used, the position is preferably between the second position from the N-terminus and the 30th position from the C-terminus (including the second position from the N-terminus and the 30th position from the C-terminus). In other words, the reactive amino acid residue is preferably included in the random sequence. Therefore, the preferred position of the random sequence can be set from the same range as the preferred positions of the reactive amino acid residues.

[0043] The number of the random sequences in the peptide may be 1 or may be 2 or more. The upper limit of the number of the random sequences is not particularly limited and may be appropriately selected depending on the purpose, but is preferably 10 or less. The number of amino acid residues per random sequence is not particularly limited and can be appropriately selected depending on the purpose; for example, it can be 1 or more and 30 or less. The longer each random sequence is and the greater the number of random sequences is, the greater the diversity of the peptide library can be.

[0044] The peptide may further include a sequence for purifying the polypeptide chain, such as a FLAG (registered trademark) sequence or a poly-His sequence, a sequence that is selectively cleaved by a protease or the like, a spacer sequence, and the like.

[0045] The number of amino acid residues in the peptide is not particularly limited and can be appropriately selected depending on the purpose, and can be, for example, 10 or more and 5,000 or less. The lower limit of the number of amino acid residues in the peptide is preferably not less than 150, more preferably not less than 200. The upper limit of the number of amino acid residues in the peptide is preferably not more than 800, more preferably not more than 600, particularly preferably not more than 500. The lower limit and upper limit can be selected in appropriate combination.

[0046] The method for synthesizing the peptide is not particularly limited, and any known method can be appropriately selected.

[0047] <Linker molecule> The linker molecule is the above-mentioned linker molecule for producing a cell membrane-permeable cyclic peptide of the present invention. The linker molecule is introduced into the peptide by reacting a cyclizing group on the linker molecule with a reactive amino acid residue on the peptide, during which the structure of the cyclizing group changes.

[0048] <Other configurations> Other components in the peptide complex are not particularly limited as long as they do not impair the effects of the present invention and can be appropriately selected depending on the purpose, and examples thereof include luminescent substances such as fluorescent substances, dyes, radioactive substances, drugs, toxins, nucleic acids, amino acids, sugars, lipids, various polymers, etc. These may be used alone or in combination of two or more types. The fluorescent substance is not particularly limited and can be appropriately selected depending on the purpose. Examples of the fluorescent substance include fluorescent dyes such as fluoresceins, rhodamines, coumarins, pyrenes, and cyanines. The other components can be bound to the above-mentioned peptides, for example, directly or via a linking group.

[0049] (Method of Producing Peptide Conjugate) The method for producing a peptide conjugate of the present invention includes at least a linker molecule introducing step of introducing the linker molecule of the present invention into a peptide, and may further include other steps as necessary.

[0050] <Linker molecule introduction step> The linker molecule introducing step is a step of introducing (hereinafter also referred to as "binding", "inserting" or "linking") the linker molecule of the present invention into a peptide. The linker molecule introduction step can simultaneously cyclize the peptide and impart cell membrane permeability to the peptide. In the linker molecule introduction step, it is sufficient that the linker molecule is introduced into at least one peptide in the reaction product, but it is preferable that the linker molecule is introduced into all of the peptides.

[0051] -peptide- The peptide is the same as that described in the <Peptide> section of the above (Peptide complex). The peptide may be in the form of a peptide library.

[0052] -introduction- The method of introduction is not particularly limited and can be appropriately selected depending on the purpose. For example, a method of reacting the linker molecule with the peptide in the presence of a reducing agent can be mentioned. The reducing agent is not particularly limited and can be appropriately selected depending on the purpose. Examples of the reducing agent include tris(2-carboxyethyl)phosphine hydrochloride. The reaction conditions such as temperature and time are not particularly limited and can be appropriately selected depending on the purpose.

[0053] <Other processes> The other steps are not particularly limited as long as they do not impair the effects of the present invention and can be appropriately selected depending on the purpose. For example, there can be mentioned a step of purifying the peptide complex using preparative HPLC.

[0054] The method for confirming whether the obtained peptide complex has the desired structure is not particularly limited, and any known analytical method can be appropriately selected, such as mass spectrometry, proton nuclear magnetic resonance spectroscopy, carbon-13 nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, and infrared spectroscopy.

[0055] (Peptide Library) The peptide library of the present invention contains at least the peptide complex of the present invention, and further contains other components as necessary. The peptide library may consist solely of the peptide complex of the present invention, or may contain non-cyclized peptides.

[0056] The peptide library can be produced in the same manner as described above (Method for producing peptide complexes).

[0057] (Method of screening for functional peptides) The method for screening functional peptides of the present invention includes at least a step of screening functional peptides using the peptide library of the present invention, and may further include other steps as necessary. The screening method is not particularly limited as long as the peptide library of the present invention is used, and a known method can be appropriately selected. For example, a method of mixing a desired target substance with the peptide library, selecting a bound peptide complex (e.g., RD ​​complex), dissociating RNA from the RD complex, preparing DNA from the RNA, amplifying it, transcribing it into mRNA, and preparing an RD complex library again, repeating the steps, and screening for a functional peptide having affinity for the target substance, can be mentioned. EXAMPLES

[0058] The present invention will be described in more detail below by way of examples, but the present invention is not limited to these examples in any way.

[0059] (Preparation Example 1: Synthesis of Peptides) The peptide having the following sequence was synthesized on Rink Amide resin (0.2 mmol / g) by microwave-assisted solid-phase synthesis. FITC-Ahx-Cys-Gly-Ser-Gly-Leu-Ala-Ser-Pro-Asn-Gly-Tyr-Cys-NH2 [In the above sequences, "FITC" stands for fluorescein isothiocyanate, and "Ahx" stands for 6-aminohexanoic acid.]

[0060] The resin on which the peptide was formed was immersed in trifluoroacetic acid (TFA) / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol (92.5 / 2.5 / 2.5 / 2.5 (volume ratio)) for 3 hours to cleave the peptide from the resin. The resulting peptide was purified by reverse phase HPLC and lyophilized to obtain a peptide having the above sequence (hereinafter sometimes referred to as "P"; see the structural formula below). The identification data of the peptide P by electrospray ionization mass spectrometry (ESI-MS) was as follows: ESI-MS C 72 H 92N 16 O 22 O3 calculated value (M+2H+) 815.295, measured value 814.67 [ka]

[0061] (Example 1: Synthesis of linker molecule G3-DCX for producing cell membrane-permeable cyclic peptide) <Synthesis of Compound C1> Compound C1 represented by the following structural formula was synthesized by a method similar to that described in US Pat. No. 7,862,807. In addition, "Boc" in the structural formula represents a "tert-butoxycarbonyl group." [ka]

[0062] <Synthesis of compound C3> [ka]

[0063] According to the above reaction scheme, compound C3 represented by the above structural formula was synthesized. Specifically, a methylene chloride solution (15 mL) of compound C1 (500 mg, 0.42 mmol) was cooled to 0 ° C., and compound C2 (Tokyo Chemical Industry Co., Ltd., product number A2293) (117.5 mg, 0.504 mmol), 1-hydroxybenzotriazole (HOBT) (85.1 mg, 0.630 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC / HCl) (120 mg, 0.630 mmol) were added and stirred for 17 hours at 25 ° C. H2O (20 mL) was added thereto, extraction was performed with methylene chloride (30 mL x 3), and the organic layer was washed with 1N hydrochloric acid (20 mL x 2), saturated sodium bicarbonate water (20 mL x 2), and water (20 mL x 2). The mixture was dried over Na2SO4, filtered and concentrated, and the resulting residue was purified by silica gel chromatography (methanol (MeOH) / CH2Cl2 = 1 / 10) to obtain compound C3 as a white solid (533 mg, 0.379 mmol, yield 90%). The identification data of the compound C3 by 1H NMR was as follows. 1H NMR(CDCl3): δ 11.4(s, 3H), 8.58(t, 3 J HH =6.0Hz, 3H), 7.69(t, 3 J HH =5.0Hz, 3H), 6.84(s, 1H), 3.89(s, 2H), 3.71-3.65(m, 22H), 3.56-3.53(m, 6H), 3.42-3.38(m, 8H), 2.43(t, 3 J HH =6.0Hz, 6H), 1.49(s, 27H), 1.48(s, 27H)

[0064] <Synthesis of compound DBXA> Sodium hydride (132 mg, 3.02 mmol) was suspended in tetrahydrofuran (THF) (6 mL) and cooled to 0°C. 2-Propyn-1-ol (0.165 mL, 2.80 mmol) was added thereto and stirred at 0°C for 30 minutes. 1,3,5-Tris(bromomethyl)benzene (1.00 g, 2.80 mmol) was added and stirred at 25°C for 20 hours. Ethyl acetate (100 mL) was added, and the mixture was washed with water (100 mL) and saturated saline (100 mL), and the organic layer was washed with magnesium sulfate. The residue obtained by filtration and concentration was purified by preparative thin layer chromatography (PTLC) (methylene chloride / hexane = 1 / 2) to obtain the compound DBXA (416.6 mg, 1.25 mmol, yield 45%) represented by the following structural formula as a pale yellow oil. The identification data of the compound DBXA by 1H NMR was as follows. 1H NMR(CDCl3): δ 7.29(s, 1H), 7.26(s, 2H), 4.53(s, 2H), 4.40(s, 4H), 4.15( 4 J HH =2.5Hz, 2H), 2.43( 4 J HH =2.0Hz, 1H) [ka]

[0065] <Synthesis of compound C4> [ka]

[0066] According to the above reaction scheme, compound C4 represented by the above structural formula was synthesized. Specifically, a solution consisting of compound C3 (533 mg, 0.379 mmol), compound DBXA (188 mg, 0.568 mmol), and THF (40 mL) was bubbled with nitrogen gas to create a nitrogen atmosphere. An aqueous solution of copper sulfate (200 mM; 1.89 mL, 0.379 mmol) and an aqueous solution of sodium ascorbate (100 mM; 7.58 mL, 0.758 mmol) were added thereto, and the mixture was stirred at 25 ° C for 3 hours. Water was added to the reaction solution, and extraction was performed with methylene chloride (3 times with 20 mL), and the organic layer was dried with Na2SO4, filtered and concentrated. The resulting residue was purified by silica gel chromatography (MeOH / CH2Cl2 = 1 / 20) to obtain compound C4 as a white solid (497 mg, 0.286 mmol, yield 76%). The identification data of the compound C4 by 1H NMR was as follows. 1H NMR(CDCl3): δ 8.58(t, 3 J HH =5.5Hz, 3H), 7.77(s, 1H), 7.74(t, 3 J HH =5.0Hz, 3H), 7.34(s, 1H), 7.32(s, 2H), 6.82(s, 1H), 4.69(s, 2H), 4.58(s, 2H), 4.56(t, 3 J HH =5.0Hz, 2H), 4.47(s, 4H), 3.89(t, 3 J HH =5.0Hz, 2H), 3.87(s, 2H), 3.69(t, 3 J HH =6.0Hz, 6H), 3.66(s, 6H), 3.61(s, 8H), 3.56-3.52(m, 6H), 3.41-3.38(m, 6H), 2.42(t, 3 J HH =6.0Hz, 6H), 1.49(s, 27H), 1.48(s, 27H)

[0067] <Synthesis of the linker molecule G3-DCX for the preparation of cell membrane-permeable cyclic peptides> [ka]

[0068] According to the above reaction scheme, the linker molecule G3-DCX for preparing a cell membrane-permeable cyclic peptide represented by the above structural formula was synthesized. Specifically, compound C4 (497 mg, 0.286 mmol) and 4N hydrochloric acid dioxane solution (30 mL) were mixed and stirred at 25°C for 46 hours. After the reaction, a white solid precipitated. The supernatant was removed to obtain a crude product of linker molecule G3-DCX for producing cell membrane-permeable cyclic peptides. The crude product was purified by preparative HPLC (high performance liquid chromatography), and linker molecule G3-DCX for producing cell membrane-permeable cyclic peptides was obtained as a white solid (116 mg, 0.110 mmol, yield 54%). The identification data of the compound G3-DCX by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) was as follows: MALDI-TOF MS 24 H 51 N 14 O8 calculated value (M+H+) 1047.502, measured value 1047.953

[0069] Example 2: Synthesis of peptide conjugate G3-DCX-P [ka]

[0070] According to the above reaction scheme, the peptide complex G3-DCX-P represented by the above structural formula was synthesized. Specifically, peptide P (4.8 mg, 3.0 μmol) synthesized in Preparation Example 1 was dissolved in 50 mM ammonium bicarbonate buffer (1.0 mL), tris(2-carboxyethyl)phosphine (TCEP) (0.94 mg, 3.3 μmol) was added, and the mixture was stirred at 25° C. for 1 hour. A solution consisting of the linker molecule G3-DCX (4.7 mg, 4.5 μmol) for producing a cell membrane-permeable cyclic peptide synthesized in Example 1 and water (500 μL) was added thereto, and the mixture was stirred at 25° C. for 20 hours. The reaction solution was purified by preparative HPLC to obtain a cyclic peptide (peptide complex G3-DCX-P) (1.01 mg, 0.388 μmol, yield 13%). The analytical conditions for the preparative HPLC are as follows: The analytical chart of peptide P synthesized in Preparation Example 1 is shown in FIG. 1, and the analytical chart of the cyclic peptide (peptide complex G3-DCX-P) is shown in FIG. [Analysis conditions] Column: SUPELCO C18 4.7mm×250mm Detection wavelength: 220nm Flow rate: 1.0mL / min Solvent: A = 0.1% TFA in water, B = 0.1% TFA in acetonitrile Gradient: Solvent B concentration (%) = 0% → 100% (0 min → 20 min) Furthermore, the identification data of the peptide complex G3-DCX-P by MALDI-TOF MS was as follows: MALDI-TOF MS 114 H 163 N 32 O 33 O3 calculated value (M+H+) 2604.12, measured value 2603.575

[0071] (Test Example 1: Cell Membrane Permeability Evaluation) HeLa cells (Human cervix adenocarcinoma cells) were cultured for 2 hours under conditions of 5% (v / v) CO2 and 37°C in a cell culture medium containing 2 μM of the peptide synthesized in Preparation Example 1 or the peptide complex synthesized in Example 2. FluoroBrite D-MEM (ThermoFisher) (supplemented with 10% (v / v) FCS (fetal calf serum) and 10% (v / v) GlutaMax (ThermoFisher)) was used for culturing HeLa cells. Next, the cell surface was washed with D-PBS(-) (containing heparin (20 units / mL)), and the cells were collected and suspended in D-PBS(-) (containing 0.5% (v / v) BSA (bovine serum albumin), 200 mM EDTA (ethylenediaminetetraacetic acid), and 0.2% (v / v) propidium iodide (Sigma-Aldrich)), and the fluorescence intensity was measured using a flow cytometer (BD FACS AriaIII). In the flow cytometry analysis, the fluorescence intensity mode was calculated for the live cell population excluding propidium iodide-positive dead cells. The results are shown in Table 1.

[0072] [Table 1]

[0073] As shown in Table 1, after 2 hours of culture, the peptide synthesized in Preparation Example 1 hardly permeated the membrane, whereas the peptide complex synthesized in Example 2 permeated the membrane. This demonstrated that the introduction of the linker molecule G3-DCX for preparing a cell membrane-permeable cyclic peptide into the peptide resulted in the cyclization of the peptide and the acquisition of cell membrane permeability at the same time.

[0074] (Example 3: Preparation of a peptide library containing peptide complexes having peptides cyclized with a linker molecule G3-DCX for preparing cell membrane-permeable cyclic peptides) <(1) Preparation of RNA library> In this section (1), we will discuss random sequence (NNK) by the NNK method. 10[wherein N represents A, U, G, or C, K represents G or U, and NNK corresponds to all codons]. 12 A method for preparing an RNA library with the above diversity will now be described.

[0075] To prepare this RNA library, template DNA (base sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2) was used. Specifically, a 5' fragment was prepared using a reaction solution having the composition shown in Table 2-1 and a plasmid as template DNA in the PCR cycles shown in Table 2-2. In Table 2-1, 5FragF_150409 is the forward primer (SEQ ID NO: 4), and Ma5frag_withoutHis_R150310 is the reverse primer (SEQ ID NO: 5).

[0076] [Table 2-1]

[0077] [Table 2-2]

[0078] Next, a 3' fragment of the template DNA was prepared using a reaction solution having the composition shown in Table 2-3 and the PCR cycle shown in Table 2-4. In Table 2-3, A1MaNNK10_withoutHis_150531 is a forward primer (SEQ ID NO: 6), and 3FragR_150409 is a reverse primer (SEQ ID NO: 7).

[0079] [Table 2-3]

[0080] [Table 2-4]

[0081] Next, overlap extension PCR was performed using a reaction solution having the composition shown in Table 2-5 and the PCR cycle shown in Table 2-6 to ligate the 5' and 3' fragments and amplify the full length to obtain template DNA. In Table 2-5, X to Z are each 1×10 12 The 5' and 3' fragments were used, and H2O was added to the reaction solution to adjust the total volume to 60 μL. 5FFnew_150409 is a forward primer (SEQ ID NO: 8), and 3F-Rnew_150409 is a reverse primer (SEQ ID NO: 9).

[0082] [Table 2-5]

[0083] [Table 2-6]

[0084] The obtained DNA was used as a template in a reaction solution having the composition shown in Table 2-7, and reacted at 37° C. for 2 hours to obtain 10 12 An RNA library containing the above mRNAs was obtained. As shown in Figure 3, the mRNAs contained in this library have, from the 5' end, a FLAG (registered trademark) site, cysteine, a random sequence, cysteine, a TEV protease site, and a spacer sequence, and do not have a stop codon.

[0085] [Table 2-7]

[0086] <(2) Construction of ribosome display (RD) complex library> The RD complex was prepared using a reconstituted cell-free protein synthesis kit ("PURE frex (registered trademark)" manufactured by Gene Frontier) and the above RNA library. This RD complex reaction solution was mixed with anti-FLAG (registered trademark) M2 antibody-conjugated agarose beads (manufactured by Sigma-Aldrich, 20 μL), and stirred at 4°C for 60 minutes. The anti-FLAG M2 antibody-conjugated agarose beads to which the RD complex having the FLAG sequence in the peptide portion was selectively bound were collected.

[0087] <(3) Preparation of a ribosome display complex library incorporating the linker molecule G3-DCX for generating cell membrane-permeable cyclic peptides> After diluting the agarose beads collected in (2) above to 80 μL, 10 mM tris(2-carboxyethyl)phosphine hydrochloride (4 μL) (final concentration 0.5 mM) as a reducing agent and 200 mM G3-DCX (the linker molecule for generating cell membrane-permeable cyclic peptides synthesized in Example 1) (0.8 μL) (final concentration 2 mM) were added, and the cyclization reaction was allowed to proceed overnight at 4°C. After the cyclization reaction, the RD complex was eluted from the agarose beads by adding the FLAG peptide, and a ribosome display complex library incorporating the linker molecule G3-DCX for generating cell membrane-permeable cyclic peptides was obtained.

[0088] (Example 4: Preparation of an RD complex containing a peptide complex having a peptide cyclized with the linker molecule G3-DCX for generating cell membrane-permeable cyclic peptides) <Preparation of RNA> An mRNA was prepared in the same manner as in Example 3, except that the random sequence in the mRNA prepared in Example 3 (the portion of “(NNK)” located between cysteine residues (C) in FIG. 3) was replaced with the nucleotide sequence encoding the amino acid sequence “LYRSLPAWRYL”. 10 ” was replaced with the nucleotide sequence encoding the amino acid sequence “LYRSLPAWRYL”.

[0089] <Preparation of the RD complex> An RD complex was prepared in the same manner as in Example 3, except that the mRNA prepared in <Preparation of RNA> above was used.

[0090] <Preparation of a ribosome display complex incorporating the linker molecule G3-DCX for generating cell membrane-permeable cyclic peptides> An RD complex introduced with a linker molecule G3-DCX for producing a cell membrane-permeable cyclic peptide was obtained in the same manner as in Example 3, except that the RD complex prepared in <Preparation of the RD complex> was used.

[0091] Agarose beads were separated and removed from the reaction solution containing the RD complex, and phosphate-buffered saline (pH 7.5, 100 μL) not containing Mg 2+ was added to dissociate the RD complex, and the resulting peptide was cleaved with TEV protease to obtain peptide fragments. Peptide fragments were also obtained in the same manner for the RD complex into which the linker molecule G3-DCX for producing a cell membrane-permeable cyclic peptide was not introduced. The results of measuring the molecular weights of the obtained peptides by MALDI-TOF MS are shown in FIG. 4. In FIG. 4, the upper row shows the measurement results of the peptide fragments obtained when the linker molecule G3-DCX for producing a cell membrane-permeable cyclic peptide was not introduced, and the lower row shows the measurement results of the peptide fragments obtained when the linker molecule G3-DCX for producing a cell membrane-permeable cyclic peptide was introduced. As shown in FIG. 4, it was confirmed that a cyclized peptide (black arrow in FIG. 4) was obtained by introducing the linker molecule G3-DCX for producing a cell membrane-permeable cyclic peptide. The white arrows in FIG. 4 indicate non-cyclized peptides.

[0092] (Example 5: Preparation of an RD complex containing a peptide complex having a peptide cyclized with a linker molecule G3-DCX for producing a cell membrane-permeable cyclic peptide) A ribosome display complex introduced with mRNA, an RD complex, and a linker molecule G3-DCX for producing a cell membrane-permeable cyclic peptide was prepared in the same manner as in Example 4, except that "LYRSLPAWRYL" in the amino acid sequence in Example 4 was changed to "PLFPWPSLWHR". Also, peptide fragments were obtained from the obtained RD complex in the same manner as in Example 4, and their molecular weights were measured by MALDI-TOF MS. The results are shown in FIG. 5. In FIG. 5, the upper part shows the measurement results of peptide fragments obtained when the linker molecule G3-DCX for producing cell membrane-permeable cyclic peptides was not introduced, and the lower part shows the measurement results of peptide fragments obtained when the linker molecule G3-DCX for producing cell membrane-permeable cyclic peptides was introduced. As shown in FIG. 5, it was confirmed that a cyclized peptide (black arrow in FIG. 5) was obtained by introducing the linker molecule G3-DCX for producing cell membrane-permeable cyclic peptides. The open arrow in FIG. 5 indicates a peptide that was not cyclized.

[0093] (Example 6: Preparation of RD complex containing a peptide complex having a peptide cyclized with a linker molecule G3-DCX for preparing a cell membrane-permeable cyclic peptide) A ribosome display complex incorporating mRNA, an RD complex, and the linker molecule G3-DCX for producing a cell membrane-permeable cyclic peptide was prepared in the same manner as in Example 4, except that the amino acid sequence "LYRSLPAWRYL" in Example 4 was changed to "AGRWNVLWRTYTYMH." Moreover, peptide fragments were obtained from the obtained RD complex and their molecular weights were measured by MALDI-TOF MS in the same manner as in Example 4. The results are shown in FIG. In FIG. 6, the upper row shows the measurement results of peptide fragments obtained when the linker molecule G3-DCX for producing cell membrane-permeable cyclic peptides was not introduced, and the lower row shows the measurement results of peptide fragments obtained when the linker molecule G3-DCX for producing cell membrane-permeable cyclic peptides was introduced. As shown in FIG. 6, it was confirmed that a cyclized peptide (black arrow in FIG. 6) was obtained by introducing the linker molecule G3-DCX for producing cell membrane-permeable cyclic peptides. The open arrow in FIG. 6 indicates a peptide that was not cyclized.

[0094] As shown in Examples 4 to 6, it was confirmed that the cyclization reaction proceeds even when the peptide length or the amino acids constituting the peptide are different.

[0095] Examples of aspects of the present invention include the following. <1> The linker molecule for producing a cell membrane-permeable cyclic peptide is characterized by having a cell membrane-permeability imparting group and a cyclization group that cyclizes a peptide. <2> The cell membrane permeability imparting group has a basic functional group. <1> is a linker molecule according to the invention. <3> The basic functional group has a guanidino group. <2> is a linker molecule according to the invention. <4> The basic functional group has two or more guanidino groups. <3> is a linker molecule according to the invention. <5> The cell membrane permeability imparting group has a dendritic structure, and the basic functional group is introduced at the end of a branch of the dendritic structure. <2> from <4> The linker molecule according to any one of claims 1 to 5, <6> The cyclizing group is an electron withdrawing group. <1> from <5> The linker molecule according to any one of claims 1 to 5, <7> The electron withdrawing group is a halogen-containing <6> is a linker molecule according to the invention. <8> The electron withdrawing group has two or more halogens. <7> is a linker molecule according to the invention. <9> The electron-withdrawing group has two or more chlorine atoms. <8> is a linker molecule according to the invention. <10> The above compound, wherein the electron-withdrawing group is benzyl chloride which may have a substituent. <9> is a linker molecule according to the invention. <11> The above compound, wherein the electron-withdrawing group is a 3,5-bis(chloromethyl)benzyl group. <10> is a linker molecule according to the invention. <12> The cyclization of the peptide by the cyclization group is carried out by a reaction of the cyclization group with at least one group selected from the group consisting of a thiol group, an amino group, and a hydroxyl group contained in the peptide. <1> from <11> The linker molecule according to any one of claims 1 to 5, <13> The linker molecule for producing a cell membrane-permeable cyclic peptide is represented by the following structural formula: [ka] <14> A peptide and the above-mentioned compound introduced into the peptide. <1> from <13> and a linker molecule according to any one of claims 1 to 5. <15> The above <14> The peptide library is characterized by comprising the peptide complex according to claim 1. <16> The peptide <1> from <13> The present invention relates to a method for producing a peptide conjugate, the method comprising the step of introducing a linker molecule according to any one of the above items. <17> The peptide is a peptide contained in a peptide library. <16> 2. A method for producing the peptide conjugate according to claim 1. <18> The above <15> The present invention relates to a method for screening functional peptides, the method comprising the step of screening for functional peptides using the peptide library described in the above.

Claims

1. A linker molecule for producing a cell membrane-permeable cyclic peptide, comprising a cell membrane-permeability imparting group and a cyclization group for cyclizing a peptide, the linker molecule comprising: the cell membrane permeability imparting group and a cyclization group that cyclizes the peptide are linked via a linking group; the cell membrane permeability imparting group has a dendritic structure, and three guanidino groups are introduced at the ends of the branches of the dendritic structure; the cyclization group that cyclizes the peptide is a 3,5-bis(chloromethyl)benzyl group; A linker molecule for producing a cell membrane-permeable cyclic peptide, characterized by being represented by the following structural formula: 【Chemistry 1】

2. A linker molecule for producing a cell membrane-permeable cyclic peptide as described in claim 1, wherein the cyclization group that cyclizes the peptide reacts with at least one group selected from the group consisting of a thiol group, an amino group, and a hydroxyl group contained in the peptide to be cyclized by the cyclization group that cyclizes the peptide.

3. A peptide complex comprising a peptide and a linker molecule for producing a cell membrane-permeable cyclic peptide described in any one of claims 1 to 2, introduced into the peptide.

4. A peptide library characterized by having the peptide complex described in claim 3.

5. A method for producing a peptide complex, comprising the step of introducing a linker molecule for producing a cell membrane-permeable cyclic peptide described in any one of claims 1 to 2 into a peptide.

6. A method for producing a peptide complex described in claim 5, wherein the peptide is a peptide contained in a peptide library.

7. A method for screening functional peptides, comprising the step of screening functional peptides using the peptide library described in claim 4.

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