Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
  • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof

Definitions

• the present invention relates to a cell membrane penetrating peptide complex and a method for producing the same, a peptide library containing the peptide complex, and a method for screening a functional peptide using the peptide library.
• therapeutic agents for specific diseases and molecules having a high affinity for target molecules have been selected from a peptide (polypeptide) library having various amino acid sequences.
• a ribosome display method using a ribosome display complex containing a peptide chain, an mRNA molecule, and a ribosome is known (for example, a patent). Reference 1).
• the RD method if they have in vitro translation system and mRNA, only mixing them, in which very good useful which can be produced 10 12 or more peptide library in a few minutes.
• Techniques for incorporating the active ingredient into the cell include, for example, a method of fusing the amino acid sequences of a cell membrane penetrating peptide containing a large amount of basic amino acids, and a dendrimer which is a dendrimer polymer having a regularly branched structure from the center.
• the method to be used see, for example, Patent Document 2 and the like are known.
• the cell membrane permeability of the active ingredient is low in a short period of time, most of the active ingredient is metabolized before it is taken up by the cells, which may make it difficult to show the drug effect. Therefore, the uptake of the active ingredient into the cell is difficult. It is preferably performed in a short time.
• An object of the present invention is to solve the above-mentioned problems in the past and to achieve the following objects. That is, the present invention provides a peptide complex having excellent cell membrane permeability in a short period of time, a method for producing the same, a peptide library containing the peptide complex, and a method for screening a functional peptide using the peptide library. The purpose.
• the present inventors have a dendritic structure having four or more branches, and of the four or more branches, at least four branches have basic functional groups at the ends. It was found that the cell-penetrating permeability of the peptide complex in a short period of time can be remarkably enhanced by introducing the cell-penetrating-imparting group having the peptide into the peptide.
• the present invention is based on the above-mentioned findings of the present inventors, and the means for solving the above-mentioned problems are as follows. That is, ⁇ 1> A peptide and a cell membrane permeability-imparting group introduced into the peptide are included.
• the cell membrane permeability-imparting group has a dendritic structure having four or more branches, and has a dendritic structure. It is a peptide complex characterized by having a basic functional group at the end of at least four of the four or more branches.
• a step of introducing a cell membrane penetrating molecule into a peptide is included.
• a method for screening a functional peptide which comprises a step of screening a functional peptide using the peptide library according to the above ⁇ 3>.
• a peptide complex capable of solving the above-mentioned problems in the past and achieving the above object and having excellent cell membrane permeability in a short time and a method for producing the same, a peptide library containing the peptide complex, and the like. And a method for screening a functional peptide using the peptide library can be provided.
• the peptide complex of the present invention contains at least a peptide and a cell membrane permeability-imparting group introduced into the peptide, and further contains other configurations as necessary.
• the peptide is not particularly limited as long as the cell membrane permeability-imparting group can be introduced, and can be appropriately selected depending on the intended purpose.
• the type of amino acid in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, and may be a natural amino acid, an unnatural amino acid, or a D-form. It may be present or it may be L-form.
• the peptide may be a modified peptide such as a lipopeptide.
• the amino acid residue (hereinafter, may be referred to as “reactive amino acid residue”) used for introducing the cell membrane permeability-imparting group is not particularly limited and may be appropriately selected depending on the intended purpose.
• reactive amino acid residue for example, cysteine residue, lysine residue, histidine residue, tryptophan residue, tyrosine residue, serine residue, threonine residue and the like can be mentioned.
• the alcohol side chain at the amino acid residue may be used. These may be used alone or in combination of two or more.
• the number of the reactive amino acid residues in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose.
• the number of the reactive amino acid residues in the peptide is preferably 2 or more.
• the upper limit of the number of the reactive amino acid residues in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, but the number of cell membrane permeability-imparting groups that bind to the peptide as the number of reaction sites increases. 10 or less is preferable because the position and position may not be stable and it may be difficult to compare the characteristics of peptides derived from the amino acid sequence.
• the reactive amino acid residue may be separately introduced into the peptide. preferable.
• the position of the reactive amino acid residue in the peptide is not particularly limited and can be appropriately selected depending on the intended purpose.
• RNA chain an mRNA molecule, a peptide chain which is a translation thereof (hereinafter, may be referred to as “polypeptide chain”), and a ribosome display complex containing ribosome (hereinafter, referred to as “RD complex”).
• RD complex a ribosome display complex containing ribosome
• it is a portion protruding from the exit tunnel (exit tunnel) of the ribosome, and specifically, the position (N) from the second N-terminal to the 30th C-terminal. It is preferable to set it between (including the second position from the terminal and the 30th position from the C terminal) in that the modification reaction by the linker molecule can be less likely to be sterically inhibited by the ribosome.
• the position from the C-terminal the 50th position from the C-terminal is preferable, and the 100th position is more preferable.
• the position can be appropriately set according to the chain length of the peptide, and is, for example, the 2nd to 1,000th position from the N-terminal.
• the 2nd to 100th positions from the N-terminal are preferable, and the 2nd to 50th positions from the N-terminal are more preferable.
• the method for producing the RD complex is not particularly limited, and a known method can be appropriately selected. Examples thereof include the method described in International Publication No. 2017/213158. It can also be manufactured using a commercially available kit.
• the amino acid sequence of the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, but one containing a random sequence at a specific position is preferable so as to be useful as a peptide library. From such a random sequence, a useful amino acid sequence can be specified according to a predetermined purpose.
• the position of the random sequence in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose.
• the position is not particularly limited. It is preferably between the 2nd position from the N-terminal to the 30th position from the C-terminal (including the 2nd position from the N-terminal and the 30th position from the C-terminal). That is, the reactive amino acid residue is preferably contained in a random sequence. Therefore, the preferred position of the random sequence can be set from the same range as the preferred position of the reactive amino acid residue.
• the number of the random sequences in the peptide may be one or two or more.
• the upper limit of the number of the random sequences is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 10 or less.
• the number of amino acid residues per random sequence is not particularly limited and may be appropriately selected depending on the intended purpose, and may be, for example, 1 or more and 30 or less. The longer one random sequence and the larger the number of random sequences, the greater the diversity of the peptide library.
• the peptide may further contain a sequence for purifying a polypeptide chain such as a FLAG® sequence or a polyHis sequence, a sequence selectively cleaved by a protease or the like, a spacer sequence, or the like.
• a sequence for purifying a polypeptide chain such as a FLAG® sequence or a polyHis sequence, a sequence selectively cleaved by a protease or the like, a spacer sequence, or the like.
• the number of amino acid residues of the peptide is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it may be 10 or more and 5,000 or less, but the upper limit thereof is 800 or less. Is preferable, 400 or less is more preferable, and 200 or less is particularly preferable.
• the method for synthesizing the peptide is not particularly limited, and a known method can be appropriately selected.
• the cell membrane permeability-imparting group can be represented by, for example, the following general formula (1).
• ABC ⁇ ⁇ ⁇ General formula (1) In the general formula (1), "A” represents a peptide linking group, “B” represents a linking group or a single bond, and “C” represents a group having a dendritic structure.
• the peptide linking group contributes to the introduction of the cell membrane permeability-imparting group into the peptide by reacting with the reactive amino acid residue in the peptide.
• the reactive amino acid residue is an amino acid residue that reacts with the peptide linking group, may be an amino acid residue that directly reacts with the peptide linking group, or can react with the peptide linking group. It may be an amino acid residue modified to.
• the peptide-linking group is not particularly limited as long as it can be linked to the peptide, and can be appropriately selected depending on the intended purpose.
• the thiol group of the cysteine residue and the side chain amino of the lysine residue can be selected. Examples thereof include a group (-NH 2 ), a group capable of reacting with a side chain amino group (> NH) of a histidine residue or a tryptophan residue to form a bond, and the like.
• Specific examples of the peptide linking group include alkyl halide groups, activated carbonyl groups, and unsaturated hydrocarbon groups described in paragraphs [0067] to [0076] of International Publication No. 2017/213158.
• Examples thereof include an epoxy group, a sulfonyl-containing group, an isocyanate group, a thioisocyanate group, a carben generating group, a disulfide bond-containing group and a thiol group.
• a group represented by the following structural formula can be used as the peptide linking group.
• the linking group is a group that links the peptide linking group and the group having a dendritic structure.
• the structure of the linking group is not particularly limited and may be appropriately selected depending on the intended purpose.
• the cell membrane permeability-imparting group has a dendritic structure having four or more branches, and has a basic functional group at the end of at least four of the four or more branches.
• the group having a dendritic structure is a group that imparts cell membrane permeability to the peptide.
• the number of branches in the dendritic structure of the cell membrane permeability-imparting group is not particularly limited as long as it is 4 or more, and can be appropriately selected depending on the intended purpose. Examples thereof include 6 or more and 9 or more. ..
• "having 4 or more branches” means the total number of branches of the dendritic structure in the cell membrane permeability-imparting group.
• the cell membrane permeability-imparting group may have one dendritic structural unit having four or more branches, or may have one dendritic structural unit having three or less branches, and the plurality.
• the total number of branches in the dendritic structure unit may be 4 or more.
• 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 intended purpose, and may be one or two or more.
• the number of branches per dendritic structural unit is not particularly limited and may be appropriately selected depending on the intended purpose, but 3 or more is preferable.
• the structure of the branch of the dendritic structure is not particularly limited and may be appropriately selected depending on the intended purpose.
• the method for producing the group having a dendritic structure is not particularly limited and may be appropriately selected depending on the intended purpose.
• the group is produced by the method described in US Pat. No. 7,862,807. be able to.
• the basic functional group is not particularly limited and may be appropriately selected depending on the intended purpose. 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.
• the number of the basic functional groups is not particularly limited as long as it is 4 or more, and can be appropriately selected depending on the intended purpose. Examples thereof include 6 or more and 9 or more.
• the basic functional group preferably has 4 or more guanidino groups.
• the position of the basic functional group in the cell membrane permeability-imparting group is not particularly limited as long as the basic functional group is located at least at the end of four branches of the dendritic structure, and is appropriately selected according to the purpose. can do.
• the dendritic structure unit of the group having the dendritic structure include those represented by the following general formula (C).
• the general formula (C) represents a dendritic structural unit having 3 branches, and "Y" in the formula represents a basic functional group.
• the basic functional group may be formed at the ends of all branches, or may be formed at the ends of some branches.
• the cell-penetrating permeability-imparting group is introduced into the peptide by reacting the peptide-linking group in the cell-penetrating-imparting group with a reactive amino acid residue in the peptide. During the reaction, the structure of the peptide linking group changes.
• cell membrane permeability-imparting group examples include compounds represented by the following structural formulas.
• the cell membrane permeability-imparting group represented by the following structural formula has a group capable of binding to a peptide by the oximuligation method as a linking group for a peptide, and has a dendritic structure at all ends of three branches. This is an example in which two dendritic structural units having a guanidino group as a basic functional group are linked, and the peptide linking group and the dendritic group are linked via a linking group.
• "*" represents a linking site with a peptide.
• the cell membrane permeability-imparting group represented by the following structural formula has a group capable of binding to a peptide by the oximuligation method as a linking group for a peptide, and has a dendritic structure at all ends of all three branches. This is an example in which there are three dendritic structural units having a guanidino group as a basic functional group, and the peptide linking group and the group having the dendritic structure are linked via a linking group.
• "*" represents a linking site with a peptide.
• the other constitution of the peptide complex is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected depending on the intended purpose.
• a luminescent substance such as a fluorescent substance, a dye, a radioactive substance, etc.
• examples thereof include drugs, toxins, nucleic acids, amino acids, sugars, lipids, and various polymers. These may be used alone or in combination of two or more.
• the fluorescent substance is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include fluorescent dyes such as fluorescein, rhodamine, coumarin, pyrene and cyanine.
• the other constitution can be attached to the above-mentioned peptide, for example, directly or via a linking group or the like.
• the peptide complex of the present invention has excellent cell membrane permeability in a short period of time. Therefore, for example, by using a peptide complex library containing a random sequence and performing screening, a useful amino acid sequence such as excellent cell membrane permeability in a short time and high affinity for a target substance can be identified.
• the method for producing a peptide complex of the present invention includes at least an introduction step of introducing a cell membrane penetrating molecule into a peptide, and further includes other steps as necessary.
• the introduction step is a step of introducing a cell membrane penetrating molecule into a peptide (hereinafter, may be referred to as “binding”, “inserting”, or “linking”).
• the cell membrane permeability-imparting group in the above item peptide complex
• the cell membrane penetrating group may be introduced into at least one peptide in the reaction product, but it is preferable that the cell membrane penetrating group is introduced into all the peptides.
• the peptide is the same as that described in the item ⁇ Peptide> of the above (peptide complex).
• the peptide may be in the form of a peptide library.
• the cell membrane penetrating molecule is introduced into a peptide and exists as a cell membrane penetrating group in the above item (peptide complex). Therefore, the cell membrane-permeable molecule can be the same as the ⁇ cell membrane permeability-imparting group> of the above-mentioned (peptide complex).
• the method for producing the cell membrane penetrating molecule is not particularly limited and may be appropriately selected depending on the intended purpose.
• a step of forming a cell membrane penetrating group precursor and a peptide in the cell membrane penetrating group precursor For example, a step of forming a cell membrane penetrating group precursor and a peptide in the cell membrane penetrating group precursor.
• the method of introduction is not particularly limited and may be appropriately selected depending on the intended purpose.
• a method of reacting a peptide linking group in the cell membrane penetrating molecule with a reactive amino acid residue in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose.
• the conditions such as the temperature and time of the reaction are not particularly limited and may be appropriately selected depending on the intended purpose.
• the other steps are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose. Examples thereof include a peptide complex purification step.
• the method for confirming whether or not the obtained peptide complex has a desired structure is not particularly limited, and a known analysis method can be appropriately selected. For example, mass spectrometry and proton nuclear magnetic resonance spectroscopy can be selected. Analytical methods such as method, carbon-13 nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, and infrared spectroscopy can be mentioned.
• the peptide library of the present invention contains at least the peptide complex of the present invention, and further contains other configurations as required.
• the peptide library may consist only of the peptide complex of the present invention, or may contain a peptide into which the cell membrane permeability-imparting group has not been introduced.
• the peptide library can be produced in the same manner as described above (method for producing a peptide complex).
• the method for screening a functional peptide of the present invention includes at least a step of screening a functional peptide using the peptide library of the present invention, and further includes 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.
• a desired target substance and the peptide library are mixed, a bound peptide complex (for example, RD complex) is selected, RNA is dissociated from the RD complex, and DNA is prepared from the RNA.
• a bound peptide complex for example, RD complex
• RNA is dissociated from the RD complex
• DNA is prepared from the RNA. Examples thereof include a method of screening for a functional peptide having an affinity for the target substance by repeating the step of transcribing to mRNA and preparing an RD complex library again after amplification.
• the compound C3 represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound C1 (50 mg, 0.159 mmol) was dissolved in methylene chloride (5 mL) and cooled to 0 ° C.
• compounds C2 (284 mg, 0.239 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloric acid synthesized by the same method as described in US Pat. No. 7,862,807.
• Salt (EDC / HCl) 45.8 mg, 0.239 mmol
• 1-hydroxybenzotriazole (HOBT) 32.2 mg, 0.239 mmol
• the identification data of the compound C4 by 1H NMR was as follows.
• the cell membrane penetrating molecule G3 represented by the above structural formula was synthesized by the above reaction formula. Specifically, a dioxane hydrochloride solution (4N; 1.0 mL) was added to compound C4 to prepare a solution, and the mixture was stirred at 25 ° C. for 2 hours. The precipitated white solid was obtained by centrifugation and further washed with diethyl ether (3 times at 3 mL) to obtain a cell membrane penetrating molecule G3 (90 mg, quantitative yield).
• the identification data of the compound G3 by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows. MALDI-TOF MS C 24 H 50 N 14 O 8 Calculated value (M + H +) 663.401, measured value 663.402
• the resin on which the peptide was formed was used as trifluoroacetic acid (TFA) / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol (92.5 / 2.5 / 2.5 / 2.5 (92.5 / 2.5 / 2.5 / 2.5).
• TFA trifluoroacetic acid
• the peptide was excised from the resin by immersing it in (volume ratio)) for 3 hours.
• the obtained peptide (55.0 mg, 30.0 ⁇ mol) was dissolved in DMF (2.5 mL), and 1,3-dibromo-2-propanone (20 mM DMF solution; 1.5 mL, 30 ⁇ mol) and N-methylmorpholine ( 10 mM DMF solution; 6.0 mL, 60 ⁇ mol) was added and stirred at 25 ° C. for 1 hour. Diethyl ether (100 mL) was added thereto to remove the supernatant, and the residue was washed with diethyl ether (100 mL) to obtain compound C5 as a white solid (54.6 mg, 28.9 ⁇ mol, yield 97%). ..
• CMLYIVPYFSVGC in the structural formula of compound C5 represents the amino acid sequence of the peptide.
• the identification data of the compound C5 by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows. MALDI-TOF MS C 94 H 128 N 15 O 20 S 3 + Calculated value (M + H +) 1882.862, Measured value 1883.140
• the compound C6 represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound C5 (30 mg, 0.0159 mmol), cell membrane penetrating molecule G3 (52.7 mg, 0.0796 mmol), DMF (0.5 mL) and water (0.05 mL) were mixed and 25 ° C. Was stirred for 24 hours. Diethyl ether (5 mL) was added and centrifugation was performed to remove the supernatant. The crude product of compound C6 was obtained by washing the residue with diethyl ether (3 times at 3 mL). Compound C6 (3.8 mg, 0.00150 mmol, yield 9%) was obtained by purification by reverse phase HPLC (high performance liquid chromatography) and lyophilization.
• HPLC high performance liquid chromatography
• the peptide complex G3-P represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound C6 and a 20% piperidine / DMF solution (1.0 mL) were mixed and stirred at 25 ° C. for 2 hours using a vortex mixer. Nitrogen gas was sprayed to volatilize the solvent. The residue was washed with diethyl ether (1 mL 3 times). Here, fluorescein isothiocyanate (FITC) (0.8 mg, 0.002 mmol), diisopropylethylamine (iPr 2 EtN) (0.0022 mL, 0.0124 mmol) and DMF (0.5 mL) are mixed and 20 at 25 ° C. Stirred for hours.
• FITC fluorescein isothiocyanate
• iPr 2 EtN diisopropylethylamine
• DMF 0.5 mL
• the resin on which the peptide was formed was used as trifluoroacetic acid (TFA) / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol (92.5 / 2.5 / 2.5 / 2.5 (92.5 / 2.5 / 2.5 / 2.5).
• TFA trifluoroacetic acid
• the peptide was excised from the resin by immersing it in (volume ratio)) for 3 hours.
• the solvent was distilled off by spraying nitrogen gas, and the residue was washed with diethyl ether (10 mL) to obtain compound C7 represented by the following structural formula as a white solid (quantitative yield).
• the compound C9 represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound C8 (74 mg, 25.5 mmol) and a 20% piperidine / DMF solution (1.0 mL) were mixed and stirred at 25 ° C. for 1 hour. The solvent was volatilized by blowing nitrogen gas onto the reaction solution to obtain a crude compound C9 product as a colorless transparent oil.
• the cell membrane penetrating molecule G6 represented by the above structural formula was synthesized by the above reaction formula. Specifically, a dioxane hydrochloride solution (4N; 1.0 mL) was added to the crude compound C9 product to prepare a solution, and the mixture was stirred at 25 ° C. for 2 hours. The precipitated white solid was obtained by centrifugation and further washed with diethyl ether (3 times at 1 mL) to obtain the cell membrane permeable molecule G6 (117 mg, quantitative yield) as a white solid.
• the compound C10 represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound C5 (30 mg, 0.0159 mmol), cell membrane penetrating molecule G6 (117 mg, 0.0796 mmol), DMF (0.5 mL) and water (0.05 mL) were mixed and 92 at 25 ° C. Stirred for hours. Diethyl ether (5 mL) was added and centrifugation was performed to remove the supernatant. The crude product of compound C10 was obtained by washing the residue with diethyl ether (3 times at 3 mL). Compound C10 (4.7 mg, 0.00141 mmol, yield 9%) was obtained as a white solid by purification by reverse phase HPLC and lyophilization.
• the peptide complex G6-P represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound C10 and a 20% piperidine / DMF solution (1.0 mL) were mixed and stirred at 25 ° C. for 2 hours using a vortex mixer. Nitrogen gas was sprayed to volatilize the solvent. The residue was washed with diethyl ether (1 mL 3 times). Here, FITC (0.8 mg, 0.002 mmol), diisopropylethylamine (0.0022 mL, 0.0124 mmol) and DMF (0.5 mL) were mixed, and the mixture was stirred at 25 ° C. for 24 hours.
• the resin on which the peptide was formed was used as trifluoroacetic acid (TFA) / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol (92.5 / 2.5 / 2.5 / 2.5 (92.5 / 2.5 / 2.5 / 2.5).
• TFA trifluoroacetic acid
• the peptide was excised from the resin by immersing it in (volume ratio)) for 3 hours.
• the solvent was distilled off by spraying nitrogen gas, and the residue was washed with diethyl ether (10 mL) to obtain compound C11 represented by the following structural formula as a white solid (quantitative yield).
• the compound C12 represented by the above structural formula was synthesized by the above reaction formula. Specifically, methylene chloride (10 mL) was added to compound C11 (50 mg, 0.073 mmol) to prepare a solution, which was cooled to 0 ° C. To this, compound C2 (391 mg, 0.329 mmol), EDC / HCl (63.0 mg, 0.329 mmol) and HOBT (44.1 mg, 0.329 mmol) were added in this order, and the mixture was stirred at 0 ° C. for 14 hours. Water (30 mL) was added and the organic layer was separated. The aqueous layer was further extracted with methylene chloride (twice at 30 mL) and the organic layer was mixed.
• the compound C13 represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound C12 (93 mg, 22.1 mmol) and a 20% piperidine / DMF solution (1.0 mL) were mixed to prepare a solution, and the mixture was stirred at 25 ° C. for 30 minutes. Nitrogen gas was blown onto the reaction solution to volatilize the solvent. The obtained residue was washed with hexane (3 times at 5 mL) to obtain compound C13 (101 mg, quantitative yield) as a white solid.
• the cell membrane penetrating molecule G9 represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound C13 and a dioxane hydrochloride solution (4N; 1.0 mL) were mixed to prepare a solution, and the mixture was stirred at 25 ° C. for 2 hours. The precipitated white solid was obtained by centrifugation and further washed with diethyl ether (3 times at 5 mL) to obtain the cell membrane permeable molecule G9 (148 mg, quantitative yield) as a white solid.
• the compound C14 represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound C5 (28 mg, 0.0148 mmol), cell membrane penetrating molecule G9 (161 mg, 0.074 mmol), DMF (0.5 mL) and water (0.05 mL) were mixed to prepare a solution. After stirring at 25 ° C. for 12 hours using a vortex mixer, nitrogen gas was blown to volatilize the solvent. The residue was washed with diethyl ether (3 times at 5 mL) to give compound C14 crude product. The compound C14 (6.6 mg, 0.00163 mmol, yield 11%) was obtained as a white solid by purification by reverse phase HPLC and lyophilization.
• the peptide complex G9-P represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound C14 and a 20% piperidine / DMF solution (1 mL) were mixed and stirred at 25 ° C. for 2 hours using a vortex mixer. Nitrogen gas was sprayed to volatilize the solvent. The residue was washed with diethyl ether (1 mL 3 times). Here, FITC (0.8 mg, 0.002 mmol), diisopropylethylamine (0.0022 mL, 0.0124 mmol) and DMF (0.5 mL) were mixed, and the mixture was stirred at 25 ° C. for 60 hours.
• HeLa cells Human cervix adenocarcinoma cell
• a cell culture medium containing 2 ⁇ M of the peptide complex synthesized in Examples 1 and 2 or Comparative Example 1 under the conditions of 5% (v / v) CO 2 , 37 ° C. Cultured for hours.
• FluroBrite D-MEM manufactured by Thermo Fisher
• FCS fetal bovine serum
• 10% (v / v) GlutaMax manufactured by Thermo Fisher
• Examples of aspects of the present invention include the following.
• a peptide and a cell membrane permeability-imparting group introduced into the peptide are included.
• the cell membrane permeability-imparting group has a dendritic structure having four or more branches, and has a dendritic structure. It is a peptide complex characterized by having a basic functional group at the end of at least four of the four or more branches.
• ⁇ 2> The peptide complex according to ⁇ 1>, wherein the basic functional group has 4 or more guanidino groups.
• ⁇ 3> The peptide complex according to any one of ⁇ 1> to ⁇ 2>, wherein the number of amino acid residues of the peptide is 200 or less.
• ⁇ 4> The peptide complex according to any one of ⁇ 1> to ⁇ 3>, wherein the cell membrane permeability-imparting group has a dendritic structural unit represented by the following general formula (C).
• C general formula
• "Y" represents a basic functional group.
• the cell membrane permeability-imparting group is the peptide complex according to any one of ⁇ 1> to ⁇ 4> represented by the following structural formula.
• "*" represents a linking site with a peptide.
• the cell membrane permeability-imparting group is the peptide complex according to any one of ⁇ 1> to ⁇ 4> represented by the following structural formula.
• "*" represents a linking site with a peptide.
• a step of introducing a cell membrane penetrating molecule into a peptide is included.
• ⁇ 9> The method for producing a peptide complex according to any one of ⁇ 7> to ⁇ 8>, wherein the number of amino acid residues of the peptide is 200 or less.
• ⁇ 12> The method for producing a peptide complex according to any one of ⁇ 7> to ⁇ 10>, wherein the cell membrane permeability-imparting group is represented by the following structural formula.
• "*" represents a linking site with a peptide.
• ⁇ 13> A peptide library comprising the peptide complex according to any one of ⁇ 1> to ⁇ 6>.
• ⁇ 14> A method for screening a functional peptide, which comprises a step of screening a functional peptide using the peptide library according to ⁇ 13>.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Biophysics (AREA)
• Genetics & Genomics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Analytical Chemistry (AREA)
• Gastroenterology & Hepatology (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (3)

Applications Claiming Priority (2)

Publications (1)

Family

ID=72611797

Family Applications (1)

Country Status (4)

Cited By (2)

Citations (3)

Family Cites Families (1)

• 2020
  • 2020-02-14 WO PCT/JP2020/005762 patent/WO2020195303A1/ja not_active Ceased
  • 2020-02-14 US US17/442,225 patent/US20220162259A1/en not_active Abandoned
  • 2020-02-14 JP JP2021508239A patent/JP7529217B2/ja active Active
  • 2020-02-14 EP EP20776861.5A patent/EP3950700A4/en not_active Withdrawn

Patent Citations (3)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events