Cell membrane permeable peptide for coupling

Abstract

The present invention provides a cell membrane permeable peptide for coupling or the like, which is represented by general formula (P1) [in general formula (P1), R101 represents a 2-pyridyl group which may have a substituent; a1 is a cell membrane permeable peptide; z12 is a divalent organic group; and q2 is 0 or 1], the cell membrane permeable peptide is a peptide in which two or more amino acids are bonded by a peptide bond, and at least one of the amino acid residues constituting the peptide has a C1-30 alkyl group substituted by at least two fluorine atoms in a side chain, or a group having 1-5 ether-bonded oxygen atoms between carbon atoms of a C2-30 alkyl group substituted by at least two fluorine atoms. R101-S-S-(Z12) q2-A1 (P1)

Description

Technical Field

[0001] The present invention relates to a cell membrane permeable peptide for coupling with excellent cell membrane permeability, which can efficiently introduce nucleic acid into cells by binding to nucleic acid.

[0002] This application claims priority based on Japanese Patent Application No. 2022-170448, filed in Japan on October 25, 2022, the contents of which are incorporated herein by reference. Background Art

[0003] In recent years, the research of nucleic acid drugs using oligonucleotides has been carried out. Nucleic acid drugs have the advantages of high specificity to target molecules, few side effects. However, the cell membrane permeability of nucleic acid drugs is low, and it is difficult to reach the target molecules present in the cell. Particularly, since siRNA is double-stranded, therefore compared with antisense RNA, molecular weight and negative charge are all larger, and cell membrane permeability is lower than antisense RNA, and it is necessary to utilize carriers to carry out drug delivery. As drug delivery agents, there are known drug delivery agents (patent documentation 1) using lipid nanoparticles, drug delivery agents (patent documentation 2) using cationic polymer nanoparticles. However, most of the efficiency of cell membrane permeability, toxicity issues need to be improved.

[0004] On the other hand, compounds containing polyfluorinated structures are known to be stable in vivo, have low toxicity, and are excellent in cellular uptake and elimination from endosomes (Non-Patent Document 1). Research has also been conducted to utilize this property to introduce polyfluorinated structures into oligonucleotides and peptide nucleic acids as moieties that have cell membrane permeability (Patent Documents 3 and 4).

[0005] In addition, research has been conducted on introducing polyfluorinated structures into peptides. For example, it has been reported that dendrimer peptides using lysine, obtained by perfluoroacylation of the side chain amino group, as a constituent amino acid can be used for gene delivery (Non-Patent Document 2).

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. 2011 / 036557

[0009] Patent Document 2: International Publication No. 2017 / 212006

[0010] Patent Document 3: International Publication No. 2012 / 130941

[0011] Patent Document 4: International Publication No. 2021 / 060506

[0012] Non-patent literature

[0013] Non-patent literature 1: Zhang et al., MRS Communications, 2018, vol. 8, pp. 303-313.

[0014] Non-patent literature 2: Cai et al., ACS Applied Materials and Interfaces, 2016, vol. 8, pp. 5821-5832.

[0015] Non-patent document 3: Murayama et al., Chemistry A European Journal, 2013, vol. 19, pp. 14151-14158. Summary of the Invention

[0016] The present invention aims to provide a cell membrane permeable peptide coupled to a target nucleic acid to be introduced into a cell, which improves the cell membrane permeability of the nucleic acid without impairing the function of the nucleic acid within the cell, and a nucleic acid for transfection coupled to the cell membrane permeable peptide.

[0017] The present inventors produced peptides containing amino acid residues with fluoroalkyl groups introduced into their side chains and found that these peptides exhibited excellent cell membrane permeability. Furthermore, they discovered that by linking these peptides to a target nucleic acid (target nucleic acid) to be introduced into cells via a disulfide bond, they could impart high cell membrane permeability to the nucleic acid without impairing its intracellular function, leading to the completion of the present invention.

[0018] That is, the present invention is as follows.

[0019] [1] A cell membrane permeable peptide for conjugation, represented by the following general formula (P1):

[0020] R 101 -SS-(Z 12 )q2A 1 (P1)

[0021] [In the general formula (P1), R 101 A is a 2-pyridyl group which may have a substituent; 1 Z is a cell membrane permeable peptide; 12 is a divalent organic group; q2 is 0 or 1],

[0022] The cell membrane permeable peptide is a peptide composed of two or more amino acids bonded by peptide bonds, and at least one of the amino acid residues constituting the peptide has a C substituted with at least two fluorine atoms in its side chain. 1－30 Alkyl, or C substituted by at least 2 fluorine atoms 2－30 An alkyl group having 1 to 5 ether-bonded oxygen atoms between carbon atoms.

[0023] [2] The cell membrane permeable peptide for coupling according to [1], wherein the C 1－30 Alkyl or C substituted by at least 2 fluorine atoms 2－30 The side chain of the group having 1 to 5 ether-bonded oxygen atoms between carbon atoms of the alkyl group is a group represented by the following general formula (f-1) or (f-2),

[0024]

[0025] [Wherein, Rf P It represents a fully halogenated C containing at least 2 or more fluorine atoms. 1－10 Alkyl, or fully halogenated C 2－10 A group having 1 to 5 ether-bonded oxygen atoms between carbon atoms of the alkyl group, wherein n1 is an integer of 0 to 10, n2 is an integer of 0 to 9, and a black dot represents a bonding site].

[0026] [3] The cell membrane permeable peptide for conjugation according to [1] or [2], wherein the C 1－30 Alkyl or C substituted by at least 2 fluorine atoms 2－30 The alkyl group having 1 to 5 ether-bonded oxygen atoms between carbon atoms may be further substituted with a halogen atom other than a fluorine atom.

[0027] [4] The cell membrane-permeable peptide for conjugation according to any one of [1] to [3] above, wherein the C-terminus or N-terminus of the cell membrane-permeable peptide may be protected by a protecting group.

[0028] [5] The cell membrane-permeable peptide for conjugation according to any one of [1] to [4], wherein the Z 12 An alkylene group having 1 to 10 carbon atoms, an alkenylene group having 1 to 10 carbon atoms, an oxygen atom (-O-), a sulfur atom (-S-), -NH-, -N(CH3)-, -N(C2H5)-, -N(C3H7)-, -C(=O)-, -S(=O)2-, a polyethylene glycol group (PEG: -(C2H4O)n-), a siloxane bond, a silyl ether bond, a group obtained by removing two hydrogen atoms from a cycloalkane, a group obtained by removing two hydrogen atoms from an aromatic ring, a group obtained by removing two hydrogen atoms from a heterocycle, a sugar, or a group obtained by combining two or more of these.

[0029] [6] A method for producing a nucleic acid for transfection, comprising: binding the cell membrane-permeable peptide for coupling described in any one of [1] to [5] to a target nucleic acid to be introduced into a cell represented by the following general formula (P2) to produce a nucleic acid of the following general formula (P3),

[0030] B 1 -(Z 11 )q1-SH (P2)

[0031] [In general formula (P2), B 1 is nucleic acid; Z 11 is a divalent organic group; q1 is 0 or 1]

[0032] B 1 -(Z 11 )q1-SS-(Z 12 )q2-A 1 (P3)

[0033] [In the general formula (P3), R 101 、A 1 、Z 12 and q2 are the same as those in the above general formula (P1); B 1 、Z 11 and q1 are the same as those in the above general formula (P2)].

[0034] [7] A nucleic acid for transfection, comprising a target nucleic acid to be introduced into a cell and a cell membrane permeable peptide linked via a linker group,

[0035] The linking group comprises at least one disulfide bond,

[0036] The cell membrane permeable peptide is a peptide composed of two or more amino acids bonded via peptide bonds, wherein at least one of the amino acid residues constituting the peptide has a C substituted with at least two fluorine atoms in its side chain. 1－30 Alkyl, or C substituted by at least 2 fluorine atoms 2－30 An alkyl group having 1 to 5 ether-bonded oxygen atoms between carbon atoms.

[0037] [8] The nucleic acid for transfection according to [7], wherein the nucleic acid comprises the C 1－30 Alkyl or C substituted by at least 2 fluorine atoms 2－30 The side chain of the group having 1 to 5 ether-bonded oxygen atoms between carbon atoms of the alkyl group is a group represented by the following general formula (f-1) or (f-2),

[0038]

[0039] [Wherein, Rf P It represents a fully halogenated C containing at least 2 or more fluorine atoms. 1－10 Alkyl, or fully halogenated C 2－10 A group having 1 to 5 ether-bonded oxygen atoms between carbon atoms of the alkyl group, wherein n1 is an integer of 0 to 10, n2 is an integer of 0 to 9, and a black dot represents a bonding site].

[0040] [9] The nucleic acid for transfection according to [7] or [8], wherein the C-terminus or N-terminus of the cell membrane-permeable peptide may be protected by a protecting group.

[0041]

[10] A method for transfection of a nucleic acid, comprising bringing the nucleic acid for transfection described in any one of [7] to [9] into contact with a cell to introduce the nucleic acid into the cell.

[0042] The cell membrane-permeable peptide for coupling of the present invention comprises a cell membrane-permeable peptide portion and a linker portion having a disulfide bond. Therefore, by coupling the cell membrane-permeable peptide for coupling of the present invention to a nucleic acid to be introduced, a transfection nucleic acid can be produced in which the cell membrane-permeable peptide and the nucleic acid to be introduced are linked via a disulfide bond.

[0043] Since this transfection nucleic acid contains a cell membrane-permeable peptide, it has excellent cell membrane permeability. However, the peptide portion exists in a cleaved state within the cell. Therefore, this transfection nucleic acid has high cell membrane permeability and can fully function within the cell to the same degree as the nucleic acid before modification with the peptide, making it very useful as a nucleic acid carrier for delivery into cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This graph compares the mean fluorescence intensity of HeLa cells treated in Experimental Example 1 with a sample solution containing a FAM-modified nucleic acid (control-DNA) or a FAM-modified nucleic acid modified with a cell membrane-permeable peptide (CPP-DNA) at 37°C for 4 hours by flow cytometry. DETAILED DESCRIPTION

[0045] As used herein and in this specification, "nucleic acid" refers to molecules composed of nucleotides linked by phosphodiester bonds. These nucleotides include not only naturally occurring nucleotides such as DNA and RNA, but also artificial nucleotides that have been modified from naturally occurring nucleotides to form phosphodiester bonds with them. Examples of artificial nucleotides include nucleotides in which the side chains of natural nucleotides are modified with functional groups such as amino groups, nucleotides in which the hydroxyl group at the 2' position of the ribose backbone is substituted with a methoxy group, a fluoro group, a methoxyethyl group, or the like, phosphorothioate nucleotides (nucleotides in which the oxygen atom of the phosphate group is substituted with a sulfur atom), morpholino nucleotides (nucleotides in which ribose or deoxyribose is substituted with a morpholine ring), BNA (Bridged Nucleic Acid), HNA (Hexitol Nucleic Acid), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), TNA (Threose Nucleic Acid), GNA (Glycerol Nucleic Acid), and CeNA (Cyclohexenyl Nucleic Acid) (Non-Patent Document 3). In addition, "nucleic acid" includes any of molecules composed of only one or more natural nucleotides bonded via phosphodiester bonds, such as DNA and RNA, molecules composed of one or more natural nucleotides and one or more artificial nucleotides bonded via phosphodiester bonds, and molecules composed of only one or more artificial nucleotides bonded via phosphodiester bonds.

[0046] In the present invention and the specification of this application, "C p1－p2 "(p1 and p2 are positive integers satisfying p1<p2) refers to a group having a carbon number of p1~p2.

[0047] In the present invention and the specification of this application, "C 1－10 "Alkyl" refers to an alkyl group with 1 to 10 carbon atoms, which may be a straight chain or a branched chain. 2－10 "Alkyl" refers to an alkyl group with 2 to 10 carbon atoms, which may be a straight chain or a branched chain. 1－10 Examples of the alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, and decyl.

[0048] In the present invention and the specification of this application, "C 1－30 "Alkyl" refers to an alkyl group with 1 to 30 carbon atoms, which may be a straight chain or a branched chain. 2－30"Alkyl" refers to an alkyl group with 2 to 30 carbon atoms, which may be a straight chain or a branched chain. 1－30 Examples of the alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, triacontyl and the like.

[0049] In the present invention and the specification of this application, "C 1－6 "Alkyl" refers to an alkyl group with 1 to 6 carbon atoms, which may be a straight chain or a branched chain. 1－6 Examples of the alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, and hexyl.

[0050] In the present invention and the specification of this application, "C 6－14 "Aryl" is an aromatic hydrocarbon group having 6 to 14 carbon atoms, and is particularly preferably C 6－12 Aryl. As C 6－14 Examples of the aryl group include a phenyl group, a naphthyl group, an anthracenyl group, and a 9-fluorenyl group, and a phenyl group is particularly preferred.

[0051] In the present invention and the specification of this application, "C 6－14 "Aryl" is a group bonded to C 6－14 A group in which one or more, preferably one to three, hydrogen atoms of carbon atoms of an aryl group are replaced by another functional group. When there are two or more substituents, the substituents may be of the same type or different types. Examples of the substituent include a nitro group, a halogen atom (fluorine atom, chlorine atom, bromine atom, or iodine atom), a C 1－6 Alkyl, C 1－6 Alkoxy, and methylenedioxy (-O-CH2-O-), etc. As "substitutable C 6－14 Examples of "aryl" include phenyl, naphthyl, anthracenyl, 4-nitrophenyl, 4-methoxyphenyl, 2,4-dimethoxyphenyl, 3,4-dimethoxyphenyl, 4-methylphenyl, 2,6-dimethylphenyl, 3-chlorophenyl, 1,3-benzodiphenyl Azol-5-yl, etc.

[0052] In the present invention and the specification of this application, "C 6－14 Aryl-C 1－6 Alkyl is bonded to C 1－6 One hydrogen atom of a carbon atom in an alkyl group is replaced by a C 6－14The group obtained by aryl. 6－14 Aryl-C 1－6 C in the alkyl group 6－14 Examples of the aryl group include phenyl, naphthyl, anthracenyl, and 9-fluorenyl, with phenyl and 9-fluorenyl being particularly preferred. 6－14 Aryl-C 1－6 C in the alkyl group 1－6 Alkyl, preferably C 1－4 Alkyl. As C 6－14 Aryl-C 1－6 Examples of the alkyl group include benzyl, diphenylmethyl, triphenylmethyl, 2-phenylethyl, 9-anthrylmethyl, and 9-fluorenylmethyl.

[0053] In the present invention and the specification of this application, a "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. A "halogen atom other than a fluorine atom" refers to a chlorine atom, a bromine atom, or an iodine atom. Examples of "halogen atoms other than a fluorine atom" are preferably a chlorine atom or a bromine atom, and particularly preferably a chlorine atom.

[0054] In the present invention and the specification of this application, "C 1－6 "Alkoxy" refers to a C 1－6 An alkyl group has an oxygen atom bonded to the end of the bond. 1－6 The alkoxy group may be linear or branched. Examples of C1-6 alkoxy groups include methoxy, ethoxy, propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0055] In the present invention and this specification, "ether-bonded oxygen atoms" refer to oxygen atoms connecting carbon atoms, and do not include oxygen atoms linked in series. An alkyl group having Nc carbon atoms (Nc is an integer greater than or equal to 2) may have a maximum of Nc-1 ether-bonded oxygen atoms. Hereinafter, "alkyl groups having ether-bonded oxygen atoms between carbon atoms" may also be referred to as "ether-bonded alkyl groups."

[0056] In the present invention and the specification of this application, "C1- 30 Alkyl (the C1- 30 When the alkyl group has 2 or more carbon atoms, it may have 1 to 5 ether-bonded oxygen atoms between carbon atoms) means "a C1-C1 substituted with at least 2 fluorine atoms" 30 Alkyl, or C substituted by at least 2 fluorine atoms 2－30 An alkyl group having 1 to 5 ether-bonded oxygen atoms between carbon atoms.

[0057] In the following, "compound n" means a compound represented by formula (n).

[0058] <<Cell membrane permeable peptide for conjugation>>

[0059] The cell membrane-permeable peptide for coupling of the present invention comprises a cell membrane-permeable peptide portion and a linker portion for coupling to a nucleic acid. The linker portion comprises at least one disulfide bond. Thus, the nucleic acid coupled to the cell membrane-permeable peptide for coupling of the present invention is linked to the cell membrane-permeable peptide via the linker portion comprising at least one disulfide bond.

[0060] In the cytoplasm, there is a high concentration of glutathione (GSH) compared to the extracellular space. Therefore, in addition to significantly improving cell membrane permeability through the cell membrane permeable peptide portion, nucleic acids coupled to the cell membrane permeable peptide of the present invention also utilize GSH to reduce and cleave disulfide bonds within the cell, resulting in the nucleic acid portion and the cell membrane permeable peptide portion being separated and existing. Generally, nucleic acids modified with peptides have their structure and properties affected by the peptide, and therefore sometimes have reduced target activity within the cell compared to unmodified nucleic acids. In contrast, by coupling the target nucleic acid with the cell membrane permeable peptide of the present invention, the target nucleic acid enjoys the high cell membrane permeability provided by the cell membrane permeable peptide, while the target nucleic acid exists in a free state without modification by the cell membrane permeable peptide, thereby fully exerting the same degree of intracellular function as nucleic acids not modified with the cell membrane permeable peptide.

[0061] The cell membrane permeable peptide for coupling of the present invention is specifically represented by the following general formula (P1). In general formula (P1), A 1 is a cell membrane permeable peptide, Z 12 is a divalent organic group, q2 is 0 or 1. When q2 is 0, -(Z 12 )q2- represents a single bond. In the cell membrane permeable peptide for coupling of the present invention, the cell membrane permeable peptide portion is -A 1 , the linking group part is R 101 -S-S-(Z 12 )q2-.

[0062] R 101 -SS-(Z 12 )q2-A 1 (P1)

[0063] <Cell membrane permeable peptides>

[0064] In the general formula (P1), A 1 The cell membrane permeable peptide, i.e., the cell membrane permeable peptide constituting the cell membrane permeable peptide portion of the cell membrane permeable peptide for coupling of the present invention (hereinafter sometimes referred to as "the cell membrane permeable peptide of the present invention") is a peptide composed of two or more amino acids, and at least one of the amino acid residues constituting the peptide has a C substituted with at least two fluorine atoms in its side chain. 1－30 Alkyl. The C 1－30When the alkyl group consists of two or more carbon atoms (C 2－30 alkyl group), it may have 1 to 5 ether-bonded oxygen atoms between carbon atoms.

[0065] In the present invention and the present specification, "C 1－30 Alkyl or C substituted by at least 2 fluorine atoms 2－30 A "group having 1 to 5 ether-bonded oxygen atoms between carbon atoms of an alkyl group" may be referred to as "Rf". That is, at least one of the amino acid residues constituting the cell membrane-permeable peptide of the present invention has Rf in its side chain.

[0066] One or more hydrogen atoms bonded to carbon atoms of Rf may be further substituted by halogen atoms other than fluorine atoms. 1－30 Alkyl, preferably C 1－20 Alkyl, more preferably C 1－10 Alkyl, more preferably C 2－10 Alkyl, more preferably C 2－8 In Rf, the number of hydrogen atoms substituted with fluorine atoms is not particularly limited as long as it is 2 or more, and is, for example, preferably 3 or more, more preferably 6 or more, and even more preferably 7 or more.

[0067] Examples of Rf include trifluoromethyl, pentafluoroethyl, heptafluoropropyl, nonafluorobutyl, perfluoropentyl, perfluorohexyl, perfluoroheptyl, perfluorooctyl, perfluorononyl, perfluorodecyl, difluoromethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 1,1,2,2-tetrafluoroethyl, 1,1,2,2,3,3-hexafluoropropyl, 1,1,2,3,3,3-hexafluoropropyl, 1,1,2,2,3,3-hexafluorohexyl, 1,1,2,2,3,3-hexafluorooctyl, 1,1,2,2,3,3-hexafluorodecyl, 1,1,2,2,3,3-hexafluorooctadecyl, and 1,1,2,2,3,3-hexafluorohexacosyl.

[0068] When Rf is a group with 2 carbon atoms, Rf is preferably a group in which at least 4 hydrogen atoms bonded to a carbon atom are substituted with fluorine atoms, such as a 1,1,1-trifluoroethyl group (CF3-CH2-) or a pentafluoroethyl group. Furthermore, when Rf is a group with 3 carbon atoms, Rf is preferably a linear group. In the case of a branched group, Rf is preferably a group having 0 or 1 trifluoromethyl groups, such as a 1,1,1,3,3,3-hexafluoropropane-2-yl group ((CF3)2-CH-). When Rf is a group with 4 carbon atoms, Rf is preferably a linear group. In the case of a branched group, Rf is preferably a group in which hydrogen atoms bonded to a carbon atom constituting the alkylene moiety are substituted with fluorine atoms, or a fully fluorinated group.

[0069] As Rf, a group represented by the following general formula (f-1) or (f-2) is preferred. Here, Rf P It represents a fully halogenated C containing at least 2 or more fluorine atoms. 1－10 Alkyl. Rf P It is C 1－10 All hydrogen atoms of the alkyl group are substituted with halogen atoms, and at least two of these halogen atoms are fluorine atoms. P When the number of carbon atoms is 2 or more, it is completely halogenated. 2－10 In the case of an alkyl group, there may be 1 to 5 ether-bonded oxygen atoms between carbon atoms. In the present invention and the specification of this application, "a fully halogenated C 1－10 Alkyl (the C 1－10 The alkyl group may have an ether-bonded oxygen atom between carbon atoms when the alkyl group has 2 or more carbon atoms. 1－10 Alkyl or fully halogenated C 2－10 A group having 1 to 5 ether-bonded oxygen atoms between carbon atoms of the alkyl group. In the general formula (f-2), the two Rf P They may be the same type of groups or different types of groups.

[0070] In the following general formula (f-1) or (f-2), n1 is an integer from 0 to 10, and n2 is an integer from 0 to 9. When n1 and n2 are 0, they both represent a single bond. That is, when n1 is 0, the group represented by the general formula (f-1) is Rf P -, when n2 is 0, the group represented by the general formula (f-2) is (Rf P )2-CH-.

[0071]

[0072] When Rf is a group represented by the general formula (f-1), Rf is preferably Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, and n1 is an integer of 0 to 4, and more preferably Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, and n1 is an integer of 0 to 2, and more preferably Rf P A group in which n1 is an integer of 0 to 2 (excluding groups in which n1 is 1 and Rf P is a trifluoromethyl group).

[0073] When Rf is a group represented by the general formula (f-2), Rf is preferably Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, and n2 is an integer of 0 to 4, and more preferably Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, and n2 is an integer of 0 to 2, and more preferably Rf P A group in which n2 is an integer of 0 to 2 (excluding groups in which n2 is 0 or 1 and Rf P is a trifluoromethyl group).

[0074] As Rf, among the groups represented by the general formula (f-1) and the groups represented by the general formula (f-2), the groups represented by the general formula (f-1) are preferred, and the groups wherein n1 in the general formula (f-1) is 0 are more preferred. Among them, it is particularly preferred that n1 in the general formula (f-1) is 0 and Rf P A group in which two F atoms are bonded to the carbon atom closest to the bonding site, that is, a group in which the site adjacent to the bonding site is -CF2-.

[0075] Examples of Rf include difluoromethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 1,1,2,2-tetrafluoroethyl, 1,1,2,2,3,3-hexafluoropropyl, and 1,1,2,3,3,3-hexafluoropropyl.

[0076] As the cell membrane permeable peptide of the present invention, a peptide containing at least one amino acid residue whose side chain is Rf (Rf-containing peptide) can be mentioned. As long as at least one side chain of the amino acid residues constituting the peptide is Rf, the side chains of all amino acid residues can also be Rf. When a peptide molecule has two or more amino acid residues whose side chains are Rf, these multiple Rfs can be of the same type or different types. In addition, the amino acid residue whose side chain is Rf in the peptide can be at the N-terminus, at the C-terminus, or at a position other than the end.

[0077] The cell membrane permeable peptide of the present invention may be a peptide composed of 2 or more amino acids, and preferably a peptide composed of 3 or more amino acids. The cell membrane permeable peptide of the present invention is preferably a peptide composed of 2 to 40 amino acids, and more preferably a peptide composed of 3 to 20 amino acids.

[0078] The C-terminus or N-terminus of the cell membrane permeable peptide of the present invention that is not linked to the linker moiety may be protected by a protecting group. As the protecting group for the C-terminus, the protecting group R for the carboxyl group described below may be used.1 The groups mentioned in the preceding are preferably benzyl. In addition, as the protecting group for the N-terminus, the protecting group R of the amino group described below can be used. 2 The groups mentioned in are preferably a Boc group or an Fmoc group.

[0079] The amino acid residues in the cell membrane-permeable peptides of the present invention that do not have an Rf side chain are not particularly limited and may be α-amino acid residues, β-amino acid residues, γ-amino acid residues, or δ-amino acid residues. Furthermore, they may be L-amino acid residues or D-amino acid residues. The amino acid residues in the cell membrane-permeable peptides of the present invention that do not have an Rf side chain are preferably amino acids constituting proteins or their D-isomers, as well as amino acid residues of modified amino acids whose side chains are modified.

[0080] Examples of amino acids constituting proteins include glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, asparagine, glutamine, proline, aspartic acid, glutamic acid, lysine, arginine, and histidine. Examples of modified amino acids in which amino acids constituting proteins are modified include, for example, lysine, arginine, and histidine in which the hydrogen atoms of the amino groups in the side chains are substituted with the following R 2 The amino acid obtained by the groups mentioned in the above, the Pbf group (N-ω-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl); the hydrogen atom of the carboxyl group of the side chain of aspartic acid and glutamic acid is substituted with the following R 1 an amino acid obtained by replacing the groups exemplified in , an alkyl group such as tert-butyl; and an amino acid obtained by replacing the hydrogen atom of the thiol group of cysteine with a benzyl group.

[0081] Examples of the cell membrane permeable peptide of the present invention include tripeptides represented by the following general formula (101-1), (101-2), (102-1), or (102-2). In the general formula (101-1), (101-2), (102-1), and (102-2), R 11 and R 12 Each independently is C 1－6 Alkyl or benzyl, each independently preferably methyl or benzyl, particularly preferably R 11 is methyl, R 12 is benzyl. X is hydrogen, Fmoc or Boc. Z is C 1－6 Alkoxy, hydroxy, or amino. Z is C 1－6 In the case of an alkoxy group, a methoxy group is particularly preferred as Z. In addition, the black dot is the bonding site with the linking group portion.

[0082]

[0083] In the general formulas (101-1), (101-2), (102-1) and (102-2), Rf P , n1 and n2 are the same as those in the general formulas (f-1) and (f-2). As the group represented by the general formula (101-1), (101-2), (102-1) or (102-2), Rf is preferably P Fully fluorinated C 1－10 Alkyl, n1 or n2 is an integer from 0 to 4, more preferably Rf P is trifluoromethyl, pentafluoroethyl, heptafluoropropyl, nonafluorobutyl, perfluoropentyl, perfluorohexyl, perfluoroheptyl, or perfluorooctyl, n1 or n2 is an integer from 0 to 2, and Rf is more preferably P It is nonafluorobutyl, perfluoropentyl, perfluorohexyl, perfluoroheptyl, or perfluorooctyl, and n1 or n2 is an integer of 0-2.

[0084]

[0085] Examples of the cell membrane-permeable peptides of the present invention include tripeptides represented by the aforementioned general formula (103-1) or (103-2). In general formulas (103-1) and (103-2), Rf is the same as described above, and n3 is 1, 2, or 3. The black dots indicate the bonding sites to the linking groups of the present invention.

[0086] In the general formulas (103-1) and (103-2), R 11 、R 12 , X and Z are the same as those in the general formula (101-1) and the like. As the tripeptide of the general formula (103-1) or (103-2), R is preferably 11 and R 12 Each independently represents a methyl group or a benzyl group, and R 11 is methyl, R 12 It is benzyl.

[0087] In the general formulas (103-1) and (103-2), Rh is a hydrogen atom or C 1－6 Alkyl. Rh is C 1－6 When alkyl, as Rh, preferably C 1－3 Alkyl group, more preferably methyl or ethyl group.

[0088] In the general formulas (103-1) and (103-2), Z 2 is a 2-, 3-, or 4-valent linking group other than an alkylene group. 2 , is not particularly limited as long as it is a divalent to tetravalent group other than an alkylene group. For example, Z 2Examples include oxygen atoms (-O-), sulfur atoms (-S-), -NH-, -N(CH3)-, -N(C2H5)-, -N(C3H7)-, trivalent nitrogen atoms, -C(=O)-, -S(=O)2-, groups obtained by removing 2 to 4 hydrogen atoms from cycloalkanes, groups obtained by removing 2 to 4 hydrogen atoms from aromatic rings, groups obtained by removing 2 to 4 hydrogen atoms from heterocycles, groups obtained by combining alkylene groups with these, and combinations thereof. As aryl and heteroaryl groups, the groups listed above can be used. However, groups consisting solely of alkylene groups and groups in which the linking portion to Rf is an alkylene group are not included. As Z 2 , preferably -C(=O)-, -C(=O)-O-, -O-C(=O)-, -NH-C(=O)-O-, -O-C(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-, -S-S-, -S(=O)2-NH-, -NH-S(=O)2-, -S(=O)2-NH-S(=O)2-, -C(=O)-NH-Ph- (-Ph- is 1,4-phenylene, 1,3-phenylene, 1,5-phenylene, or 1,3,5-substituted phenyl).

[0089] The cell membrane permeable peptide of the present invention can be synthesized by general peptide synthesis methods, such as solid-phase peptide synthesis, using an Rf-containing amino acid having at least one Rf group introduced as a side chain as a raw material amino acid. The cell membrane permeable peptide of the present invention can be easily synthesized using an automatic peptide synthesizer using an amino acid having an Rf group introduced as a side chain as a raw material.

[0090] The peptide can be produced by sequentially condensing an amino acid whose amino group is protected with an amino acid whose C-terminus is bonded to a solid phase, and then releasing the peptide from the solid phase. Preferably, the amino acid raw material uses an amino acid whose amino group is protected by a Boc group or an Fmoc group. The side chain functional groups of the amino acid raw material are preferably protected by a protecting group. Examples of protecting groups for side chain functional groups include a Boc group, a triphenylmethyl group, a benzyl group, and a 2,2,5,7,8-pentamethylchroman-6-sulfonyl group (Pmc group).

[0091] Examples of peptide bond-forming condensing agents include N,N-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (WSC), and benzotriazol-1-yloxy-tris(dimethylamino) Hexafluorophosphate (BOP), benzotriazol-1-yloxytripyrrolidino Hexafluorophosphate (pyBOP), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, 1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholine carbon Hexafluorophosphate (COMU), etc. In addition, N-hydroxybenzotriazole (HOBt), ethyl (hydroxyimino)cyanoacetate (oxyma) and the above-mentioned condensing agents can also be mixed and used in a preferred ratio.

[0092] To form a peptide bond, methods for activating the carboxyl terminus can be used. Examples of activating agents include N-hydroxysuccinimide, p-nitrophenyl ester, and pentafluorophenyl ester. Examples of bases used in peptide bond formation include triethylamine and diisopropylethylamine (DIPEA). Examples of solvents used in peptide bond formation reactions include chloroform, dichloromethane (DCM), dichloroethane (DCE), acetonitrile (ACN), N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO).

[0093] The Boc group and Fmoc group, which are protecting groups for the amino terminal amino group of a peptide or amino acid, can be removed with trifluoroacetic acid or piperidine, respectively. Protecting groups for side chain functional groups of amino acid residues in a peptide can be removed with, for example, trifluoroacetic acid (TFA), hydrogen fluoride (HF), trifluoromethanesulfonic acid, and the like.

[0094] In addition, in peptide solid-phase synthesis, TFA can be used, for example, to remove a peptide or a peptide with a protective group attached to the side chain functional group of an amino acid residue from a peptide solid-phase synthesis resin. The removal of the peptide from the peptide solid-phase resin and the removal of the protective group from the side chain functional group of the amino acid residue can be performed simultaneously within the same reaction system. Alternatively, they can be performed independently. Examples of commercially available peptide solid-phase synthesis resins for peptide solid-phase synthesis include 4-hydroxymethyl-3-methoxyphenoxybutyric acid-benzhydrylamine-polystyrene resin, p-benzyloxybenzyl alcohol-polystyrene resin, and oxime resins.

[0095] The target peptide or its intermediate can be isolated and purified by various methods such as ion chromatography, gel filtration chromatography, reverse phase chromatography, normal phase chromatography, recrystallization, extraction, fractional crystallization, etc. In addition, the peptides thus obtained can be converted into their respective salts by conventional methods.

[0096] The amino or carboxyl protecting groups of the produced Rf-containing peptide may be deprotected as needed. Deprotection can be performed using conventional methods depending on the type of protecting group.

[0097] <Synthesis reaction of Rf-containing amino acids>

[0098] The Rf-containing amino acid can be produced, for example, by the following synthetic reaction.

[0099]

[0100] Specifically, Rf is preferably a group represented by the general formula (f-1) or (f-2) described later.

[0101] R 1 is a protecting group for the carboxyl group, specifically, a protecting group selected from the group represented by the following general formula (p-1), 2-(9,10-dioxo)anthracenylmethyl, benzyloxymethyl, and phenacylmethyl. 3 Can replace C6- 14 Aryl, R 4 and R 5 Each independently represents a hydrogen atom or a substituted C6- 14 In addition, black dots indicate bonding sites.

[0102]

[0103] As R 1 The protecting group of the carboxyl group represented by can be exemplified by benzyl, diphenylmethyl, triphenylmethyl, 4-nitrobenzyl, 4-methoxybenzyl, 2,4-dimethoxybenzyl, 3,4-dimethoxybenzyl, 4-methylbenzyl, 2,6-dimethylbenzyl, 3-chlorobenzyl, 9-anthrylmethyl, piperonyl, 2-(9,10-dioxo)anthrylmethyl, benzyloxymethyl, phenacylmethyl, etc. In terms of being able to be deprotected under mild conditions, R 1 Preferred are benzyl and triphenylmethyl, and more preferred are benzyl.

[0104] The production method is carried out by using an aralkyl protecting group such as benzyl or triphenylmethyl as the protecting group R of the carboxyl group. 1 This is advantageous in that R1 can be deprotected under mild conditions, and the synthesis of fluorinated amino acids and fluorinated peptides can be performed without decomposing the functional groups of the amino acids.

[0105] R 6 is a silyl protecting group. 6 , and examples thereof include trimethylsilyl (TMS) group, triethylsilyl (TES) group, triisopropylsilyl (TIPS) group, tert-butyldimethylsilyl (TBDMS) group, tert-butyldiphenylsilyl (TBDPS) group, etc. 6 It is a trimethylsilyl (TMS) group.

[0106] R 2As a protecting group of amino group. 2 , as long as it is an amino protecting group used in peptide synthesis, there is no particular limitation. As amino protecting groups, there can be mentioned carbamate protecting groups such as tert-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc). In terms of being able to be deprotected under mild conditions, R 2 Preferred is a tert-butoxycarbonyl (Boc) group or a 9-fluorenylmethyloxycarbonyl (Fmoc) group.

[0107] [Process 1]

[0108] Compound 2-2 can be obtained by reacting compound 2 with compound 8 in the presence of a metal fluoride. Compound 8 represented by Rf-R6 of general formula (8) can be synthesized in a single step from readily available Rf-I (fluoroalkyl iodide), so a wide range of Rf groups can be introduced.

[0109] As the metal fluoride, alkali metal fluorides such as cesium fluoride, lithium fluoride, and sodium fluoride can be used, and cesium fluoride is preferred.

[0110] The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include tetrahydrofuran (THF), dichloromethane (DCM), acetonitrile, benzene, toluene, diethyl ether, 1,4-dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide, with tetrahydrofuran being preferred.

[0111] The amount of compound 8 is preferably 0.5 to 10 moles relative to 1 mole of compound 2. The amount of metal fluoride is preferably 0.01 to 2 moles relative to 1 mole of compound 2. The reaction of step 1 is preferably carried out at a temperature of 10°C or less. By carrying out the reaction at a temperature of 10°C or less, compound 2-2 can be produced in a high yield. The reaction temperature is preferably -78°C to 10°C, more preferably -50°C to -10°C, and particularly preferably -40°C to -20°C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[0112] Compound 2 can be produced by diesterification of oxalic acid by a known method, or a commercially available product can be used.

[0113] [Process 1-1]

[0114] In the reaction of step 1, compound 2-1 (one side of the hydroxyl group is R 6 Protected compound), or a mixture of compound 2-2 and compound 2-1. In this case, by protecting the silyl group R 6Deprotection can yield compound 2-2.

[0115] The reaction in step 1-1 can be carried out by the same method as in step 1.

[0116] [Process 1-2]

[0117] By protecting the silyl group R 6 Deprotection can yield compound 2-2.

[0118] Deprotection can be carried out in the presence of a fluoride salt such as tetrabutylammonium fluoride (TBAF), cesium fluoride, or hydrofluoride, or an acid such as hydrochloric acid, acetic acid, or p-toluenesulfonic acid.

[0119] The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, diethyl ether, 1,4-dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide. Tetrahydrofuran is preferred. The reaction is preferably carried out with the addition of acetic acid.

[0120] The amount of the fluoride salt is preferably 0.1 to 10 moles relative to 1 mole of compound 2-1 (1 mole of the mixture when it is a mixture of compound 2-2 and compound 2-1). The amount of the acid is preferably 0.1 to 10 moles relative to 1 mole of compound 2-1 (1 mole of the mixture when it is a mixture of compound 2-2 and compound 2-1). The reaction of step 1-2 is preferably carried out at a temperature of 50°C or less. By carrying out the reaction at a temperature of 50°C or less, compound 2-2 can be produced in a high yield. The reaction temperature is preferably -80°C to 50°C, more preferably -40°C to 30°C, and particularly preferably -20°C to 30°C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[0121] [Process 2]

[0122] Compound 3 can be obtained by dehydrating compound 2-2.

[0123] The dehydration reaction can be carried out in the presence of a dehydrating agent such as phosphorus pentoxide, concentrated sulfuric acid, calcium chloride, sodium sulfate, magnesium sulfate, calcium sulfate, molecular sieves (synthetic zeolite), or silica gel. Phosphorus pentoxide is preferred as the dehydrating agent. The amount of the dehydrating agent is preferably 10 to 100 weight percent relative to 100 weight percent of compound 2-2. The dehydration reaction can be carried out by distilling compound 2-2 in the presence of the dehydrating agent. The distillation is preferably carried out at a temperature of 30°C to 150°C. If the distillation temperature is too high, compound 3 may decompose. If the distillation temperature is too low, compound 3 may not be condensed, and the recovery rate may be reduced. The distillation can be carried out under any pressure, including reduced pressure, normal pressure, or increased pressure, and can be appropriately determined so that the boiling point of compound 3 falls within the preferred temperature range described above. The pressure is preferably 0.1 mmHg to 5 atmospheres (3800 mmHg).

[0124] [Process 3]

[0125] Compound 4 can be obtained by reacting Compound 3 with Compound 9 or Compound 10.

[0126] In the general formula (9), R 2 As mentioned above, it is a protecting group for amino group. 7 、R 8 and R 9 Each independently is C 6－14 Aryl. As R 7 、R 8 or R 9 C 6－14 Aryl groups include phenyl and naphthyl. 7 、R 8 and R 9 They are phenyl.

[0127] The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include diethyl ether, tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, 1,4-dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide, with diethyl ether being preferred.

[0128] The amount of compound 9 or compound 10 is preferably 0.5 to 10 mol per 1 mol of compound 3. The reaction temperature is preferably -78°C to 100°C, more preferably 0°C to 40°C. The reaction time is preferably 1 minute to 24 hours, more preferably 10 minutes to 4 hours.

[0129] In a preferred embodiment, a carbamate-based protecting group such as tert-butoxycarbonyl or 9-fluorenylmethyloxycarbonyl is used as the protecting group R of the amino group. 2 , R 2Deprotection can suppress the decomposition and racemization of the compound and allow the synthesis of fluorinated amino acids to proceed.

[0130] [Process 4]

[0131] Compound 5 can be obtained by reducing compound 4.

[0132] The reduction reaction can be performed by a method using a reducing agent or a method of reducing in the presence of a metal catalyst.

[0133] (1) Method using a reducing agent

[0134] As the reducing agent, a borohydride reagent such as sodium borohydride, zinc borohydride, sodium cyanoborohydride, lithium triethylborohydride, lithium tri-sec-butylborohydride, potassium tri-sec-butylborohydride, lithium borohydride, or sodium triacetoxyborohydride can be used. Preferred reducing agents are sodium borohydride or zinc borohydride, with sodium borohydride being more preferred. The amount of the reducing agent is preferably 0.5 to 10 moles per 1 mole of compound 4.

[0135] The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include diethyl ether, tetrahydrofuran, hydrochlorofluorocarbons (HCFCs) (e.g., ASAHIKLIN (registered trademark) AK-225 (a mixture of 3,3-dichloro-1,1,1,2,2-pentafluoropropane and 1,3-dichloro-1,1,2,2,3-pentafluoropropane, AGC Corporation)), dichloromethane, acetonitrile, 1,4-dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide, with diethyl ether being preferred.

[0136] The reaction temperature is preferably -78° C. to 100° C., more preferably -10° C. to 40° C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[0137] (2) Reduction method in the presence of a metal catalyst

[0138] As the metal catalyst, palladium catalysts (e.g., palladium carbon, palladium hydroxide, Pearlman catalyst, Lindlar catalyst, silica gel supported palladium catalyst, alumina supported palladium catalyst, palladium oxide), nickel catalyst (e.g., Raney nickel), platinum catalyst (e.g., platinum carbon, platinum oxide, silica gel supported platinum catalyst, alumina supported platinum catalyst), rhodium catalyst (e.g., rhodium carbon, alumina supported rhodium catalyst, rhodium oxide), ruthenium catalyst (e.g., ruthenium carbon, alumina supported ruthenium catalyst, ruthenium oxide), cobalt catalyst (e.g., Raney cobalt), etc. are mentioned, preferably palladium catalyst. The amount of the metal catalyst is preferably 0.0001 to 0.1 mol, more preferably 0.0005 to 0.02 mol, relative to 1 mol of compound 4.

[0139] The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include methanol, ethanol, isopropanol, diethyl ether, tetrahydrofuran, ethyl acetate, dichloromethane, acetonitrile, 1,4-dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide.

[0140] The reduction reaction is carried out in the presence of hydrogen. The reduction reaction can be carried out at normal pressure or under pressure. The hydrogen pressure is preferably 0.5 atmospheres to 10 atmospheres. The reaction temperature is preferably 0°C to 100°C, more preferably 10°C to 50°C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[0141] [Process 5-1]

[0142] By protecting the compound 5 R 2 Deprotection can be performed to obtain compound 6-1.

[0143] Deprotection can be carried out according to the protecting group R 2 types of .

[0144] R 2 In the case of a Boc group, deprotection can be performed under acidic conditions. Examples of the acid used include trifluoroacetic acid (TFA) and hydrochloric acid. The amount of the acid is preferably 1 to 1000 mol per 1 mol of compound 5.

[0145] The reaction can be carried out in a reaction-inert solvent. Examples of the solvent include diethyl ether, tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, 1,4-dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide, with dichloromethane and N,N-dimethylformamide being preferred. An acid can also be used as the solvent. Examples of the solvent include inorganic acids such as hydrochloric acid, acetic acid, and trifluoroacetic acid, as well as organic acids, with trifluoroacetic acid being preferred. The reaction temperature is preferably -78°C to 50°C, more preferably 0°C to 40°C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[0146] R 2 In the case of an Fmoc group, deprotection can be performed under basic conditions. Examples of the base used include secondary amines such as piperidine, morpholine, and pyrrolidine. The amount of the base is preferably 1 to 100 mol per 1 mol of compound 5.

[0147] The reaction can be carried out in a reaction-inert solvent. Examples of the solvent include diethyl ether, tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, 1,4-dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide. The reaction temperature is preferably -20°C to 80°C, more preferably 0°C to 40°C. The reaction time is preferably 1 minute to 24 hours, more preferably 5 minutes to 2 hours.

[0148] [Process 6-1]

[0149] By protecting the compound 6-1 R 1 Deprotection can yield compound 7.

[0150] Deprotection can be carried out according to the protecting group R 1 R 1 In the case of benzyl, triphenylmethyl, 9-anthrylmethyl, piperonyl, 2-(9,10-dioxo)anthrylmethyl, benzyloxymethyl, or phenacylmethyl, deprotection can be performed by reduction in the presence of a metal catalyst. The reduction reaction can be performed by the same method as the reduction in the presence of a metal catalyst in step 4.

[0151] [Process 5-2]

[0152] By protecting the compound 5 R 1 Deprotection can be performed to obtain compound 6-2. Deprotection can be performed by the same method as in step 6-1.

[0153] [Process 6-2]

[0154] By protecting the compound 6-2 R 2 Deprotection can be performed to obtain Compound 7. Deprotection can be performed by the same method as in Step 5-1.

[0155] By performing asymmetric reduction of the imine (compound 4) represented by general formula (4), an optically active fluorine-containing amino acid (Rf-containing compound) can be synthesized. In the following reaction formula, the asterisk (*) indicates that the absolute configuration of the asymmetric carbon atom marked with the asterisk is S or R. In addition, Rf, R 1 and R 2 As defined above.

[0156]

[0157] In this production method, an aralkyl protecting group such as benzyl or triphenylmethyl is used as the protecting group R of the carboxyl group. 1 , which is advantageous in that R 1Deprotection allows the synthesis of Rf-containing amino acids and Rf-containing peptides while maintaining optical activity.

[0158] [Process 7]

[0159] Compound 5-1 can be obtained by performing an asymmetric reduction reaction on compound 4.

[0160] The asymmetric reduction reaction can be carried out by reducing compound 4 in the presence of an asymmetric reduction catalyst.

[0161] As the asymmetric reduction catalyst, a transition metal complex formed by coordination of a transition metal and an asymmetric ligand can be used. Examples of the transition metal include palladium, rhodium, ruthenium, iridium, nickel, cobalt, platinum, and iron. Examples of the transition metal complex include palladium complexes, rhodium complexes, ruthenium complexes, iridium complexes, and nickel complexes.

[0162] Examples of the asymmetric ligand include dpen (1,2-diphenylethylenediamine), daipen (1,1-di(4-anisyl)-2-isopropyl-1,2-ethylenediamine), and optically active phosphine ligands. Examples of the optically active phosphine ligand include 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP), 2,2'-bis(diphenylphosphino)-5,5',6,6',7,7',8,8'-octahydro-1,1'-binaphthyl (H8-BINAP), 2,2'-bis(di-p-tolylphosphino)-1,1'-binaphthyl (Tol-BINAP), 2,2'-bis[bis(3,5-dimethylphenyl)phosphino]-1,1'-binaphthyl (Xyl-BINAP), 2,2'-bis[bis(3,5-di-tert-butyl-4-methoxyphenyl)phosphino]-1,1'-binaphthyl (DTBM-BINAP), 1,2-bis(anisylphosphino)ethane (DIPAMP), 2,3-bis(diphenylphosphino)butane (CHI RAPHOS), 1-cyclohexyl-1,2-bis(diphenylphosphino)ethane (CYCPHOS), 1,2-bis(diphenylphosphino)propane (PROPHOS), 2,3-bis(diphenylphosphino)-5-norbornene (NORPHOS), 2,3-O-isopropylidene-2,3-dihydroxy-1,4-bis(diphenylphosphino)butane (DIOP), 1-[1',2-bis(diphenylphosphino)ferrocenyl]ethylamine (BPPFA), 1-[1',2-bis(diphenylphosphino)ferrocenyl]ethanol (BPPFOH), 2,4-bis(diphenylphosphino)pentane (SKEWPHOS), 1,2-bis(substituted phosphino)benzene (DuPHOS), 5,5'-bis(diphenylphosphino)-4,4'-bis-1,3-benzodione SEGPHOS, 5,5'-bis[di(3,5-xylyl)phosphino]-4,4'-bis-1,3-benzodioxadiazole oxadiazole (DM-SEGPHOS), 5,5'-bis[bis(3,5-di-tert-butyl-4-methoxyphenyl)phosphino]-4,4'-bis-1,3-benzodiazole oxazole (DTBM-SEGPHOS), 1-[2-(disubstituted phosphino) ferrocenyl] ethyl-disubstituted phosphine (Josiphos), 1-[2-(2'-disubstituted phosphinophenyl) ferrocenyl] ethyl-disubstituted phosphine (Walphos), etc.

[0163] The amount of the asymmetric reduction catalyst is preferably 0.0001 to 0.1 mol, more preferably 0.0005 to 0.02 mol, relative to 1 mol of Compound 4.

[0164] The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include methanol, ethanol, isopropanol, diethyl ether, tetrahydrofuran, ethyl acetate, dichloromethane, acetonitrile, 1,4-dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide.

[0165] The reduction reaction is carried out in the presence of hydrogen. The reduction reaction can be carried out at normal pressure or under pressure. The hydrogen pressure is preferably 0.5 to 10 atmospheres. The reaction temperature is preferably 0°C to 100°C, more preferably 10°C to 50°C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[0166] [Process 8-1]

[0167] By removing the protective group R 2 Deprotection can be performed to obtain compound 6-3. Deprotection can be performed in the same manner as in step 5-1.

[0168] [Process 9-1]

[0169] By protecting the compound 6-3 R 1 Deprotection can be performed to obtain compound 7-1. Deprotection can be performed in the same manner as in step 6-1.

[0170] [Process 8-2]

[0171] By protecting the compound 5-1 with the group R 1 Deprotection can be performed to obtain compound 6-4. Deprotection can be performed in the same manner as in step 6-1.

[0172] [Process 9-2]

[0173] By protecting the compound 6-4 R 2Deprotection can be performed to obtain compound 7-1. Deprotection can be performed in the same manner as in step 5-1.

[0174] The synthesis of optically active fluorine-containing amino acids (Rf-containing compounds) can also be carried out by the following reaction. In the following reaction formula, the asterisk indicates that the absolute configuration of the asymmetric carbon atom marked with the asterisk is S or R. In addition, Rf, R 1 and R 2 As defined above.

[0175]

[0176] [Process 10-1]

[0177] Compound 6-3 can be obtained by optical resolution of compound 6-1.

[0178] Optical resolution can be performed by a known method, for example, a method using a chiral column, a method based on crystallization, a diastereoisomer method, or the like.

[0179] (1) Method using chiral column

[0180] The racemate can be resolved into optically active forms by liquid chromatography or supercritical fluid chromatography (SFC) using a chiral column. Examples of chiral columns that can be used include CHIRALPAK (registered trademark) (Dacel Corporation) and CHIRALCEL (registered trademark) (Dacel Corporation).

[0181] (2) Crystallization-based methods

[0182] A racemic salt is formed with an optically active amine or acid, which is then induced into a crystalline diastereomeric salt by fractional crystallization. Repeated recrystallization can yield a single diastereomeric salt. If desired, the diastereomeric salt can be neutralized to yield the free, optically active form. Examples of optically active amines include strychnine, cinchonidine, cinchonine, and 1-phenylethylamine. Examples of optically active acids include camphorsulfonic acid, tartaric acid, and mandelic acid.

[0183] (3) Diastereoisomer method

[0184] A racemate is reacted with an optically active agent to obtain a mixture of diastereomers, which is then separated into individual diastereomers by fractional crystallization or chromatography. The optically active agent is removed from the resulting individual diastereomers to obtain the desired optical isomer.

[0185] [Process 11-1]

[0186] By protecting the compound 6-3 R 1Deprotection can be performed to obtain compound 7-1. Deprotection can be performed in the same manner as in step 6-1.

[0187] [Process 10-2]

[0188] Compound 6-2 can be optically resolved to obtain compound 6-4. Optical resolution can be performed by the same method as in step 10-1.

[0189] [Process 11-2]

[0190] By protecting the compound 6-4 R 2 Deprotection can be performed to obtain compound 7-1. Deprotection can be performed in the same manner as in step 5-1.

[0191] [Process 12]

[0192] Compound 7-1 can be obtained by optical resolution of compound 7. Optical resolution can be performed by the same method as in step 10-1.

[0193] <Method for producing Rf-containing peptide>

[0194] The Rf-containing peptide (cell membrane-permeable peptide of the present invention) can be produced using amino acids having Rf introduced into their side chains as raw materials. For example, the Rf-containing peptide can be produced using Compound 6-1, Compound 6-2, Compound 6-3, or Compound 6-4 as raw materials.

[0195] For example, an Rf-containing peptide can be produced by condensing compound 6-2 or 6-4 with a carboxyl-protected fluorinated amino acid, a carboxyl-protected amino acid, a C-terminally protected fluorinated peptide, or a C-terminally protected peptide. Alternatively, an Rf-containing peptide can be produced by condensing compound 6-1 or 6-3 with an amino-protected fluorinated amino acid, an amino-protected amino acid, an N-terminally protected fluorinated peptide, or an N-terminally protected peptide.

[0196] Alternatively, Compound 7 or Compound 7-1 can be protected at its amino or carboxyl group and then subjected to the same procedure to produce an Rf-containing peptide. Specifically, the amino group can be protected with a protecting group and then condensed with a carboxyl-protected fluorinated amino acid, a carboxyl-protected amino acid, a C-terminally protected fluorinated peptide, or a C-terminally protected peptide. Alternatively, the carboxyl group can be protected with a protecting group and then condensed with an amino-protected fluorinated amino acid, an amino-protected amino acid, an N-terminally protected fluorinated peptide, or an N-terminally protected peptide.

[0197] <Linking group part>

[0198] In the general formula (P1), Z 12The divalent organic group is not particularly limited, and examples thereof include an alkylene group, an alkenylene group, an oxygen atom (-O-), a sulfur atom (-S-), -NH-, -N(CH3)-, -N(C2H5)-, -N(C3H7)-, -C(=O)-, -S(=O)2-, a polyethylene glycol group (PEG: -(C2H4O)n-), a siloxane bond, a silyl ether bond, a group obtained by removing two hydrogen atoms from a cycloalkane, a group obtained by removing two hydrogen atoms from an aromatic ring, a group obtained by removing two hydrogen atoms from a heterocycle, and sugars. Examples of the aromatic ring and heterocycle include a pyrrole ring, a pyrazole ring, an imidazole ring, a triazole ring, a pyridine ring, a pyrimidine ring, a pyrazine ring, azole ring, thiazole ring, furan ring, thiophene ring, benzene ring, indole ring. As alkylene and alkenylene, they may be straight chain or branched, and may have a ring. 12 A divalent organic group or a part thereof, preferably an alkylene group having 1 to 10 carbon atoms (C1- 10 Alkylene) or alkenylene (C2- 10 Alkenylene). In addition, they can also be appropriately combined as part of the linking group of the present invention. Examples of the group formed by combining them include -C(=O)-O-, -O-C(=O)-, -NH-C(=O)-O-, -O-C(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-, -S(=O)2-NH-, -NH-S(=O)2-, -S(=O)2-NH-S(=O)2-, -C(=O)-NH-Ph- (-Ph- is 1,4-phenylene, 1,3-phenylene, 1,5-phenylene or 1,3,5-substituted phenyl), -C1- 10 Alkylene -CO-, -C2- 10 Alkenylene-CO-, -C1- 10 Alkylene-NH-, -C2- 10 Alkenylene-NH-.

[0199] In the general formula (P1), R 101 is a 2-pyridyl group which may have a substituent. The substituent that the 2-pyridyl group may have is not particularly limited as long as it does not inhibit the nucleophilic substitution reaction of the thiol derivative of the nucleic acid on the sulfur atom constituting the disulfide bond. 101 Specific examples include 2-pyridyl and 3-nitro-2-pyridyl groups which do not have a substituent.

[0200] For example, in the general formula (P1), R 101 is an unsubstituted 2-pyridyl group, q2 is 1, and Z 12 -C 1－10The compound of alkylene-CO- is a compound obtained by replacing one hydrogen atom bonded to a carbon atom in a fatty acid residue having 2 to 11 carbon atoms with a 2-pyridylthio group, or a compound having one hydrogen atom bonded to a nitrogen atom with a (2-pyridylthio)-C1- 10 Alkyl or (2-pyridylthio)-C 2－10 The compound of the amino group substituted by alkenyl is condensed with the amino group or carboxyl group of the cell membrane permeable peptide of the present invention. As the compound having 3 carbon atoms in the "compound obtained by replacing one hydrogen atom bonded to a carbon atom of a fatty acid having 2 to 11 carbon atoms with a 2-pyridylthio group", there can be mentioned 3-(2-pyridylthio) propionic acid N-succinimidyl ester. In the general formula (P1), R 101 is 3-nitro-2-pyridyl, q2 is 1 and Z 12 -C 1－10 Alkylene-NH- compounds are compounds obtained by replacing one hydrogen atom bonded to a carbon atom in a fatty acid residue having 2 to 11 carbon atoms with a 3-nitro-2-pyridylthio group, or compounds having one hydrogen atom bonded to a nitrogen atom with a (3-nitro-2-pyridylthio)-C 1－10 Alkyl or (3-nitro-2-pyridylthio)-C 2－10 Compounds containing an amino group substituted with an alkenyl group are obtained by condensing the amino group or carboxyl group of the cell membrane-permeable peptide of the present invention. Examples of compounds containing three carbon atoms in the "compound in which one hydrogen atom bonded to a carbon atom of a fatty acid having 2 to 11 carbon atoms is substituted with a 3-nitro-2-pyridylthio group" include N-succinimidyl 3-(3-nitro-2-pyridylthio) propionate.

[0201] <<Transfection Nucleic Acid>>

[0202] The transfection nucleic acid of the present invention is a compound in which a nucleic acid to be introduced into a cell and a cell membrane permeable peptide are linked via a linker containing at least one disulfide bond. As the cell membrane permeable peptide, the cell membrane permeable peptide of the present invention described above can be used.

[0203] <Nucleic acid to be introduced>

[0204] The target nucleic acid for transfection of the present invention is not particularly limited, as long as it is a nucleic acid for introduction into cells. The target nucleic acid is preferably a functional nucleic acid that exhibits a physiological activity upon incorporation into target cells within a living organism. Specifically, examples include nucleic acids for expressing proteins within cells, nucleic acids for inhibiting gene expression within cells, nucleic acids for genetic modification, nucleic acid aptamers that specifically bind to target biological molecules, and functional nucleic acids that affect the physiological functions of cells. Examples of nucleic acids for protein expression include cDNA and mRNA encoding proteins, as well as expression plasmid vectors incorporating a region encoding the target protein. Examples of nucleic acids for inhibiting gene expression include functional nucleic acids for RNA interference, such as siRNA, miRNA, shRNA, and antisense oligonucleotides, as well as RNAi vectors. Examples of nucleic acids for genetic modification include fragments of genomic DNA. Examples of functional nucleic acids that affect the physiological functions of cells include decoy nucleic acids and CpG (cytosine-phosphate-guanine) oligonucleotides. Alternatively, a functional nucleic acid may be formed by linking a functional molecule such as a fluorescent substance or a functional peptide to a nucleic acid. For example, a nucleic acid molecule in which a fluorescent substance is linked to the end of a single-stranded nucleic acid having a guanine quadruplex structure can be used as the nucleic acid to be introduced.

[0205] The nucleic acid to be introduced in the transfection nucleic acid of the present invention is not particularly limited and may be a nucleic acid containing all naturally occurring nucleotides or a nucleic acid containing some or all artificial nucleotides. Furthermore, it may be a single-stranded nucleic acid or a double-stranded nucleic acid. It may be DNA, RNA, or a chimeric nucleic acid of DNA and RNA.

[0206] <Linking group>

[0207] The linking group that links the nucleic acid to be introduced and the cell membrane permeable peptide in the nucleic acid for transfection of the present invention (hereinafter sometimes referred to as the "linking group of the present invention") is not particularly limited as long as it is an organic group that links the nucleic acid to be introduced and the cell membrane permeable peptide and has at least one disulfide bond at a position where the nucleic acid to be introduced and the cell membrane permeable peptide are separated by cleavage of the disulfide bond. The linking group of the present invention may be an organic group consisting only of a disulfide bond or a group formed by bonding a disulfide bond to another divalent or higher organic group. As other divalent organic groups, Z in the cell membrane permeable peptide for coupling of the present invention can be used. 12 The same groups are used as the divalent organic groups exemplified above.

[0208] The nucleic acid for transfection of the present invention represented by the following general formula (P3) can be produced by binding the cell membrane-permeable peptide for coupling of the present invention to the target nucleic acid to be introduced into cells represented by the following general formula (P2).

[0209] B 1 -(Z 11 )q1-SH (P2)

[0210] B 1 -(Z 11 )q1-SS-(Z 12 )q2-A 1 (P3)

[0211] In the general formula (P2), B 1 For nucleic acid, Z 11 is a divalent organic group, q1 is 0 or 1. When q1 is 0, -(Z 11 )q1- represents a single bond. As Z 11 The divalent organic group can be used in conjunction with the cell membrane permeable peptide Z in the present invention. 12 The same groups are used as the divalent organic groups exemplified above.

[0212] In the general formula (P3), R 101 、A 1 、Z 12 and q2 are the same as those in the above general formula (P1). In the general formula (P3), B 1 、Z 11 and q1 are the same as those in the above general formula (P2).

[0213] When the nucleic acid for transfection of the present invention is represented by the general formula (P3), the nucleic acid for transfection of the present invention is separated into the nucleic acid to be introduced-(Z 11 )q1-SH and HS-(Z 12 )q2-cell membrane permeable peptide. In the present invention, in order to minimize the effect on the function of the introduced nucleic acid in the cell, Z 11 An alkylene group having 1 to 6 carbon atoms is preferred. 12 , and may be a group formed by appropriately combining the organic groups listed above as other divalent organic groups.

[0214] The nucleic acid transfection method of the present invention is a method in which the transfection nucleic acid of the present invention is brought into contact with cells and introduced therein. The transfection nucleic acid of the present invention has excellent cell membrane permeability compared to nucleic acids not linked to the peptide due to the cell membrane permeability peptide. Therefore, the transfection nucleic acid of the present invention can be introduced into cells simply by contacting the cells, even without using conventional transfection reagents such as Lipofectamine. When the target cells into which the transfection nucleic acid of the present invention is introduced are cultured cells, the transfection nucleic acid can be introduced into the cells simply by adding the transfection nucleic acid to a culture medium for culturing. In addition, for animal tissues, for example, by spraying or applying a solution containing the transfection nucleic acid of the present invention onto the surface of the tissue, the transfection nucleic acid can be introduced into the cells constituting the tissue. Therefore, for example, by producing the transfection nucleic acid of the present invention by using a functional nucleic acid that exhibits a certain physiological activity when taken into target cells in an organism as the target nucleic acid for introduction, the efficiency of the uptake of the functional nucleic acid into the target cells can be improved. That is, by using the transfection nucleic acid of the present invention, a drug delivery system for delivering nucleic acid drugs into cells can be easily constructed.

[0215] Example

[0216] Hereinafter, the present invention will be described based on examples, but the present invention is not limited to these examples.

[0217] The NMR instrument used in the analysis of the examples and comparative examples was JNM-ECZ400S (400 MHz) manufactured by JEOL Ltd. 1 In HNMR, tetramethylsilane was set to 0 PPM. 19 In FNMR, the reference value of C6F6 is set to -162 PPM.

[0218] In this specification, the following abbreviations are used.

[0219] Fmoc: 9-fluorenylmethyloxycarbonyl

[0220] C6F 13 :1,1,2,2,3,3,4,4,5,5,6,6,6-tridecafluorohexyl

[0221] COMU: 1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinium hexafluorophosphate (CAS RN: 1075198-30-9)

[0222] Oxyma: Ethyl (hydroxyimino)cyanoacetate (CAS RN: 3849-21-6)

[0223] DIPEA: diisopropylethylamine

[0224]

[0225] Cell Culture

[0226] In the following experiments, cell culture was performed as follows.

[0227] HeLa cells (Riken Cell Bank) were cultured in a humidified atmosphere (5% CO2) at 37°C using low-glucose Dulbecco's modified Eagle's medium (D-MEM, manufactured by Fujifilm Wako Co., Ltd.) supplemented with 10% FBS and 0.5% penicillin / streptomycin.

[0228] 24 hours before the introduction of nucleic acid into cells, to reach 1.0 × 10 4 The cells were seeded in a 96-well culture plate (manufactured by VIOLAMO) at a cell / well format and allowed to adhere and proliferate (pre-culture).

[0229] [Production Example 1]

[0230] Containing RF (C6) amino acid (Fmoc-Asp (C6F 13 Synthesis of )-OH)

[0231]

[0232] 4-(Perfluorohexyl)aniline was synthesized according to the method described in Org. Lett., 2019, 21, 6481.

[0233] Separately, in a dry, 25 mL, two-necked eggplant flask, 150 mg (0.3 mmol) of 4-(perfluorohexyl)aniline and 130 mg (0.33 mmol, 1.1 equivalents) of Fmoc-Asp-OAll were dissolved in 6 mL of dichloromethane, and 141 mg (0.33 mmol, 1.1 equivalents) of COMU, 47 mg (0.33 mmol, 1.1 equivalents) of oxyma, and 85.3 mg (0.66 mmol, 2.2 equivalents) of DIPEA were added, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was then quenched with HCl (1 N) and extracted three times with dichloromethane. The combined organic phases were concentrated under reduced pressure, diluted with ethyl acetate, and washed with HCl (1 N), a saturated aqueous sodium bicarbonate solution, and saturated brine. The washed organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain Fmoc-Asp (C6F 13 The crude product was dissolved in acetone and then purified by reprecipitation using hexane to obtain pure Fmoc-Asp(C6F 13 )-OAll was obtained as a white solid (173 mg, 0.19 mmol, yield 64.9%).

[0234] [Production Example 2]

[0235] Tripeptide H-Ala-Asp(C6F 13 Synthesis of )-Phe-NH2

[0236]

[0237] Rink amide resin (39 mg, 0.025 μmol) and DMF (2 mL) were added to a solid-phase synthesis tube and stirred at room temperature for 30 minutes to swell the resin. A 20% piperidine / DMF solution (2 mL) was then added to the tube and stirred for 3 minutes, followed by washing with DMF (2 mL) three times. A 20% piperidine / DMF solution (2 mL) was added again and stirred for 12 minutes to deprotect the Fmoc group. Next, Fmoc-Phe-OH (39 mg, 0.10 mmol, 4.0 equiv), COMU (43 mg, 0.10 mmol, 4.0 equiv), oxyma (14 mg, 0.10 mmol, 4.0 equiv), and DIPEA (26 mg, 0.20 mmol, 8.0 equiv) were added and stirred at room temperature for 2 hours to carry out the amino acid condensation reaction. The reaction mixture was then washed with DMF (2 mL) three times, and Fmoc deprotection was performed using the same method as above. Fmoc-Asp(C6F 13 )-OH (84 mg, 0.10 mmol, 4.0 equivalents) and Fmoc-Ala-OH (31 mg, 0.10 mmol, 4.0 equivalents) were subjected to the same operation. As a subsequent post-treatment, a mixed solution for excision (TFA: triisopropylsilane: water = 95:2.5:2.5 (volume ratio)) (2 mL) was added and stirred at room temperature for 2 hours to excise from the resin to obtain a product containing H-Ala-Asp (C6F 13 The crude product was vacuum dried and then purified by reverse phase chromatography (ACN / water / TFA = 75:25:0.1 to 99:1:0.1 (volume ratio)) to obtain H-Ala-Asp (C6F 13 )-Phe-NH2 (5 mg, 5 μmol, yield 20%).

[0238] [Example 1]

[0239] Preparation of fluorescently labeled nucleic acid linked to the tripeptide H-Ala-Asp (C6F 13 )-Phe-NH2 and the resulting compound.

[0240]

[0241] SPDP (3-(2-pyridylthio) propionate N-succinimidyl ester) and tripeptide H-Ala-Asp (C6F 13 )-Phe-NH2 (compound (1)) is condensed with the amino group to obtain compound (2).

[0242] Specifically, compound (1) (1 mg), SPDP (1 mg), ACN (200 μL) and DIPEA (1 μL) were added and stirred at room temperature for 18 hours. The obtained compound was passed through a 0.45 μm filter to remove insoluble matter to obtain a crude product.

[0243] The crude product of the obtained compound (2) was separated and prepared using reverse phase HPLC (60% ACN / 40% H2O-100% ACN (volume ratio)), and the purified compound (2) was confirmed by LC-MS.

[0244] ESI-MS

[0245] [M+H]+: m / z calcd for 941.18, found: 941.51

[0246] The entire amount of the purified compound (2) was used to carry out a condensation reaction with a FAM-modified DNA with a thiol group derived therefrom. As the FAM-modified DNA with a thiol group derived therefrom, a DNA consisting of a base sequence of SEQ ID NO: 1 (TTTTTCAGTTGACCATATA) was used, a DNA having a thiol hexamethylene group (a group obtained by replacing the hydrogen atom of the carbon atom bonded to the terminal of the n-hexamethylene group with a thiol group) linked to the 5' end and FAM linked to the 3' end was used.

[0247]

[0248] The entire amount of the purified compound (2) was mixed with FAM-modified DNA (10 nmol) with a thiol group derived therefrom, dissolved in a mixed solvent of water and DMSO (water / DMSO = 1:1 (volume ratio)), and stirred at room temperature for 2 days to react. The resulting reaction mixture was purified by reverse phase chromatography (ACN containing TEAA (10 mM) / water containing TEAA (10 mM) = 10:75 to 100:0 (volume ratio)) to obtain compound (3) (yield 40.0% calculated using a fluorometer).

[0249] MALDI-TOF MS[M]: m / z calcd for 7463, found: 7638

[0250] [Test Example 1]

[0251] Compound (3) synthesized in Example 1 (comprising the tripeptide H-Ala-Asp(C6F 13 )-Phe-NH2-modified FAM-modified DNA: hereinafter referred to as "CPP-DNA"), and the efficiency of its uptake into cells was investigated. As a comparison, FAM-modified DNA (hereinafter referred to as "control-DNA") was used.

[0252] <Preparation of sample solution>

[0253] Control DNA or CPP DNA was prepared in OPTI-MEM medium to a concentration of 4 μM as a sample solution. OPTI-MEM liquid medium was used for blank measurement.

[0254] <Evaluation of cell membrane permeability by flow cytometry>

[0255] After pre-incubation at 37°C for 24 hours, the culture medium of HeLa cells was replaced with the sample solution, and cell membrane permeability was evaluated after incubation at 37°C for 4 hours. After incubation, the cell surface was washed three times with PBS (physiological saline solution), and the cells were detached and recovered using trypsin-EDTA 0.05% (Gibco). The recovered cells were then loaded onto a flow cytometer (guava easyCyte (trademark) 8) and the green 2 fluorescence (448 nm) of the fluorescent dye fluorescein (FAM) introduced into the synthetic nucleic acid was measured.

[0256] The results of the mean fluorescence intensity of each cell measured by flow cytometry are shown in Figure 1 In the figure, "control-DNA" shows the results of cells in which control-DNA was added to the culture medium, and "CPP-DNA" shows the results of cells in which FAM-modified nucleic acid (CPP-DNA) was added to the culture medium. Figure 1 As shown, cells treated with CPP-DNA showed a 1.6-fold increase in fluorescence intensity compared to cells treated with control-DNA. A T-test indicated that the observed difference in CPP-DNA and control-DNA uptake was statistically significant.

[0257] Industrial applicability

[0258] The present invention provides a transfection nucleic acid comprising a target nucleic acid to be introduced into a cell, bound to a cell-permeable peptide via a disulfide bond. The transfection nucleic acid of the present invention is efficiently introduced into the cell via the cell-permeable peptide, but is separated from the cell-permeable peptide within the cell, thereby fully realizing the function of the nucleic acid. Therefore, the transfection nucleic acid of the present invention is expected to be used in the medical field as a vector for delivering bioactive substances, such as gene drugs used in genetic modification, into target cells.

Claims

1. A cell membrane permeable peptide for coupling, represented by the following general formula (P1): R 101 -SS-(Z 12 )q2-A 1 (P1) In the general formula (P1), R 101 A is a 2-pyridyl group which may have a substituent, 1 is a cell membrane permeable peptide, Z 12 is a divalent organic group, q2 is 0 or 1, The cell membrane permeable peptide is a peptide composed of two or more amino acids bonded by peptide bonds, and at least one of the amino acid residues constituting the peptide has a C substituted with at least two fluorine atoms in its side chain. 1－30 Alkyl, or C substituted by at least 2 fluorine atoms 2－30 An alkyl group having 1 to 5 ether-bonded oxygen atoms between carbon atoms.

2. The cell membrane permeable peptide for coupling according to claim 1, wherein Having at least two fluorine atoms substituted C 1－30 Alkyl or C substituted by at least 2 fluorine atoms 2－30 The side chain of the group having 1 to 5 ether-bonded oxygen atoms between carbon atoms of the alkyl group is a group represented by the following general formula (f-1) or (f-2), Where Rf P It represents a fully halogenated C containing at least 2 or more fluorine atoms. 1－10 Alkyl, or fully halogenated C 2－10 The alkyl group has 1 to 5 ether-bonded oxygen atoms between carbon atoms, n1 is an integer of 0 to 10, n2 is an integer of 0 to 9, and a black dot represents a bonding site.

3. The cell membrane permeable peptide for coupling according to claim 1, wherein The C 1－30 Alkyl or C substituted by at least 2 fluorine atoms 2－30 The alkyl group having 1 to 5 ether-bonded oxygen atoms between carbon atoms may be further substituted with a halogen atom other than a fluorine atom.

4. The cell membrane permeable peptide for coupling according to claim 1, wherein The C-terminus or N-terminus of the cell membrane-permeable peptide may be protected by a protecting group.

5. The cell membrane permeable peptide for coupling according to claim 1, wherein The Z 12 An alkylene group having 1 to 10 carbon atoms, an alkenylene group having 1 to 10 carbon atoms, an oxygen atom (-O-), a sulfur atom (-S-), -NH-, -N(CH3)-, -N(C2H5)-, -N(C3H7)-, -C(=O)-, -S(=O)2-, a polyethylene glycol group (PEG: -(C2H4O)n-), a siloxane bond, a silyl ether bond, a group obtained by removing two hydrogen atoms from a cycloalkane, a group obtained by removing two hydrogen atoms from an aromatic ring, a group obtained by removing two hydrogen atoms from a heterocycle, a sugar, or a group obtained by combining two or more of these.

6. A method for producing a nucleic acid for transfection, comprising binding a cell membrane-permeable peptide for coupling according to any one of claims 1 to 5 to a target nucleic acid to be introduced into a cell represented by the following general formula (P2) to produce a nucleic acid represented by the following general formula (P3). B 1 -(Z 11 )q1-SH (P2) In the general formula (P2), B 1 For nucleic acid, Z 11 is a divalent organic group, q1 is 0 or 1, B 1 -(Z 11 )q1-S-S-(Z 12 )q2-A 1 (P3) In the general formula (P3), R 101 、A 1 、Z 12 and q2 are the same as those in the general formula (P1), B 1 、Z 11 and q1 are the same as those in the general formula (P2).

7. A nucleic acid for transfection, comprising a target nucleic acid to be introduced into a cell and a cell membrane permeable peptide linked via a linker group, The linking group comprises at least one disulfide bond, The cell membrane permeable peptide is a peptide composed of two or more amino acids bonded via peptide bonds, wherein at least one of the amino acid residues constituting the peptide has a C substituted with at least two fluorine atoms in its side chain. 1－30 Alkyl, or C substituted by at least 2 fluorine atoms 2－30 An alkyl group having 1 to 5 ether-bonded oxygen atoms between carbon atoms.

8. The nucleic acid for transfection according to claim 7, wherein Having at least two fluorine atoms substituted C 1－30 Alkyl or C substituted by at least 2 fluorine atoms 2－30 The side chain of the group having 1 to 5 ether-bonded oxygen atoms between carbon atoms of the alkyl group is a group represented by the following general formula (f-1) or (f-2), Where Rf P It represents a fully halogenated C containing at least 2 or more fluorine atoms. 1－10 Alkyl, or fully halogenated C 2－10 The alkyl group has 1 to 5 ether-bonded oxygen atoms between carbon atoms, n1 is an integer of 0 to 10, n2 is an integer of 0 to 9, and a black dot represents a bonding site.

9. The nucleic acid for transfection according to claim 7, wherein The C-terminus or N-terminus of the cell membrane-permeable peptide may be protected by a protecting group.

10. A method for nucleic acid transfection, comprising bringing the nucleic acid for transfection according to any one of claims 7 to 9 into contact with cells to introduce the nucleic acid therein.

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