JP2023125435A - Novel carrier molecule that delivers cell membrane-impermeable substance to cytoplasm of animal cell - Google Patents

Abstract

To provide a technique of delivering a cell membrane-impermeable substance to a cytoplasm of an animal cell.SOLUTION: There is provided a compound expressed by the following formula (1) (in the formula (1), X1 denotes a hydrogen atom, an acetyl group or -L2-R2, X2 denotes -NH2, -OH, or -L2-R2, at least one of X1 and X2 is -L2-R2, L1 and L2 denote, each independently, a divalent hydrocarbon group having a carbon number 1 to 50 in which one or a plurality of -C- may be substituted for -O-, -CO-, -NH-, -S-, or -S-S-, R1 denotes a prenyl group having an isoprene unit number 1 to 5, a saturated or unsaturated acyl group having a carbon number 10 to 30, or a saturated or unsaturated alkyl group having a carbon number 10 to 30, R2 denotes a bonding functional group or a monovalent group derived from a cell membrane-impermeable substance having a molecular weight 100 to 5000, m and n denote an integer of 0 to 20, and p and q denote an integer of 1 to 4). [formula 1]SELECTED DRAWING: None

Description

The present invention relates to novel carrier molecules that deliver substances that are impermeable to cell membranes into the cytoplasm of animal cells.

In recent years, it has been pointed out that the field of synthetic small molecule drugs has reached an impasse, and the focus of drug development has shifted to biopharmaceuticals including antibody drugs. Under these circumstances, peptide drugs are expected to serve as a next-generation drug platform to overcome the problems of small-molecule drugs.

However, peptides generally do not have cell membrane permeability, which is a major bottleneck in peptide drug discovery targeting intracellular proteins. Therefore, there is a need to develop a general-purpose technique that can introduce peptides into cells.

Up to now, several techniques for introducing peptides into cells have been developed, but in most cases the introduction into cells is via endocytosis (see, for example, Non-Patent Document 1). When a peptide is introduced into cells via endocytosis, the peptide is encapsulated within endosomes and cannot effectively exert its medicinal efficacy.

On the other hand, according to the pyrene butyrate method (see, for example, Non-Patent Document 2), peptides can be delivered to the cytoplasm. However, this approach requires pretreatment of cells with high concentrations of another compound.

Futaki S., Membrane-permeable Arginine-Rich Peptides and the Translocation Mechanisms, Adv Drug Deliv Rev., 57 (4), 547-548, 2005. Takeuchi T., et al., Direct and Rapid Cytosolic Delivery Using Cell-Penetrating Peptides Mediated by Pyrenebutyrate, ACS Chem Biol., 1 (5),299-303, 2006.

Thus, a general-purpose technique for directly delivering substances that do not permeate cell membranes, such as peptides, to the cytoplasm has not yet been established. Therefore, an object of the present invention is to provide a technique for delivering a cell membrane-impermeable substance to the cytoplasm of animal cells.

［式（１）中、Ｘ^１は水素原子、アセチル基又は－Ｌ^２－Ｒ^２を表し、Ｘ^２は－ＮＨ_２、－ＯＨ又は－Ｌ^２－Ｒ^２を表し、Ｘ^１とＸ^２の少なくとも一方は－Ｌ^２－Ｒ^２であり、Ｌ^１及びＬ^２は、それぞれ独立に、１又は複数の－Ｃ－が、－Ｏ－、－ＣＯ－、－ＮＨ－、－Ｓ－又は－Ｓ－Ｓ－に置換されていてもよい炭素数１～５０の２価の炭化水素基を表し、Ｒ^１は、イソプレン単位数１～５のプレニル基、炭素数１０～３０の飽和若しくは不飽和のアシル基又は炭素数１０～３０の飽和若しくは不飽和のアルキル基を表し、Ｒ^２は、結合性官能基、又は、分子量１００～５０００の細胞膜非透過性の物質から誘導された１価の基を表し、ｍ、ｎは、それぞれ独立に０～２０の整数を表し、ｐ、ｑは、それぞれ独立に１～４の整数を表す。］
［２］前記式（１）中のＬ^２が、－Ｓ－Ｓ－基を含む、［１］に記載の化合物。
［３］前記式（１）におけるｍ及びｎの合計が５以上である、［１］又は［２］に記載の化合物。
［４］前記式（１）におけるＸ^１が－Ｌ^２－Ｒ^２であり、ｎが７以下であるか、又は、前記式（１）におけるＸ^２が－Ｌ^２－Ｒ^２であり、ｍが７以下である、［１］～［３］のいずれかに記載の化合物。
［５］細胞に接触させた場合に、前記細胞の細胞膜を透過して細胞内に導入される、［１］～［４］のいずれかに記載の化合物。
［６］前記分子量１００～５０００の細胞膜非透過性の物質が、ペプチドである、［１］～［５］のいずれかに記載の化合物。
［７］分子量１００～５０００の細胞膜非透過性の物質を細胞透過性の物質に改変する方法であって、前記細胞膜非透過性の物質に下記式（２）で表される基又は下記式（２’）で表される基を結合させる工程を含む方法。

［式（２）及び式（２’）中、Ｌ^１及びＬ^２は、それぞれ独立に、１又は複数の－Ｃ－が、－Ｏ－、－ＣＯ－、－ＮＨ－、－Ｓ－又は－Ｓ－Ｓ－に置換されていてもよい炭素数１～５０の２価の炭化水素基を表し、Ｒ^１は、イソプレン単位数１～５のプレニル基、炭素数１０～３０の飽和若しくは不飽和のアシル基又は炭素数１０～３０の飽和若しくは不飽和のアルキル基を表し、ｍ、ｎは、それぞれ独立に０～２０の整数を表し、ｐ、ｑは、それぞれ独立に１～４の整数を表す。また、式（２）中、Ｘ^３は、－ＮＨ_２又は－ＯＨを表し、式（２’）中、Ｘ^４は、水素原子又はアセチル基を表す。］
［８］分子量１００～５０００の細胞膜非透過性の物質を対象細胞の細胞質に導入する方法であって、下記式（２）で表される基又は下記式（２’）で表される基が結合した前記物質を前記対象細胞に接触させる工程を含む方法。

［式（２）及び式（２’）中、Ｌ^１及びＬ^２は、それぞれ独立に、１又は複数の－Ｃ－が、－Ｏ－、－ＣＯ－、－ＮＨ－、－Ｓ－又は－Ｓ－Ｓ－に置換されていてもよい炭素数１～５０の２価の炭化水素基を表し、Ｒ^１は、イソプレン単位数１～５のプレニル基、炭素数１０～３０の飽和若しくは不飽和のアシル基又は炭素数１０～３０の飽和若しくは不飽和のアルキル基を表し、ｍ、ｎは、それぞれ独立に０～２０の整数を表し、ｐ、ｑは、それぞれ独立に１～４の整数を表す。また、式（２）中、Ｘ^３は、－ＮＨ_２又は－ＯＨを表し、式（２’）中、Ｘ^４は、水素原子又はアセチル基を表す。］ The present invention includes the following aspects.
[1] A compound represented by the following formula (1).

[In formula (1), X ¹ represents a hydrogen atom, an acetyl group, or -L ² -R ² , X ² represents -NH ₂ , -OH or -L ² -R ² , and the combination of X ¹ and X ² At least one is -L ² -R ² , and L ¹ and L ² each independently represent one or more -C-, -O-, -CO-, -NH-, -S- or -S Represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be substituted with -S-, R ¹ is a prenyl group having 1 to 5 isoprene units, or a saturated or unsaturated group having 10 to 30 carbon atoms. It represents an acyl group or a saturated or unsaturated alkyl group having 10 to 30 carbon atoms, and R ² represents a binding functional group or a monovalent group derived from a cell membrane-impermeable substance with a molecular weight of 100 to 5000. where m and n each independently represent an integer of 0 to 20, and p and q each independently represent an integer of 1 to 4. ]
[2] The compound according to [1], wherein L ² in the formula (1) contains a -SS- group.
[3] The compound according to [1] or [2], wherein the sum of m and n in formula (1) is 5 or more.
[4] X ¹ in the formula (1) is -L ² -R ² and n is 7 or less, or X ² in the formula (1) is -L ² -R ² and m is 7 or less, the compound according to any one of [1] to [3].
[5] The compound according to any one of [1] to [4], which when brought into contact with a cell, penetrates the cell membrane of the cell and is introduced into the cell.
[6] The compound according to any one of [1] to [5], wherein the cell membrane-impermeable substance having a molecular weight of 100 to 5,000 is a peptide.
[7] A method for modifying a cell membrane-impermeable substance with a molecular weight of 100 to 5,000 into a cell-permeable substance, the method comprising modifying the cell membrane-impermeable substance to a group represented by the following formula (2) or the following formula ( A method comprising the step of bonding a group represented by 2').

[In formula (2) and formula (2'), L ¹ and L ² each independently represent one or more -C-, -O-, -CO-, -NH-, -S- or - Represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be substituted with SS-, R ¹ is a prenyl group having 1 to 5 isoprene units, or a saturated or unsaturated group having 10 to 30 carbon atoms. represents an acyl group or a saturated or unsaturated alkyl group having 10 to 30 carbon atoms, m and n each independently represent an integer of 0 to 20, and p and q each independently represent an integer of 1 to 4. represent. Further, in formula (2), X ³ represents -NH ₂ or -OH, and in formula (2'), X ⁴ represents a hydrogen atom or an acetyl group. ]
[8] A method of introducing a cell membrane-impermeable substance with a molecular weight of 100 to 5000 into the cytoplasm of a target cell, in which a group represented by the following formula (2) or a group represented by the following formula (2') is A method comprising the step of bringing the bound substance into contact with the target cell.

[In formula (2) and formula (2'), L ¹ and L ² each independently represent one or more -C-, -O-, -CO-, -NH-, -S- or - Represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be substituted with SS-, R ¹ is a prenyl group having 1 to 5 isoprene units, or a saturated or unsaturated group having 10 to 30 carbon atoms. represents an acyl group or a saturated or unsaturated alkyl group having 10 to 30 carbon atoms, m and n each independently represent an integer of 0 to 20, and p and q each independently represent an integer of 1 to 4. represent. Further, in formula (2), X ³ represents -NH ₂ or -OH, and in formula (2'), X ⁴ represents a hydrogen atom or an acetyl group. ]

According to the present invention, it is possible to provide a technique for delivering a cell membrane-impermeable substance to the cytoplasm of an animal cell.

FIG. 1(a) is a schematic diagram showing the structure of SH2-EGFP used in Experimental Example 3. FIGS. 1(b) and 1(c) are confocal laser micrographs showing the results of Experimental Example 3. FIG. 1 is a schematic diagram illustrating an experimental system for cell membrane localization. FIG. 3(a) is a schematic diagram showing the structure of scFv GCN4-sfGFP used in Experimental Example 5. FIGS. 3(b) and 3(c) are confocal laser micrographs showing the results of Experimental Example 5. FIG. 4(a) is a schematic diagram showing the structure of 15F11-mEGFP used in Experimental Example 7. FIGS. 4(b) and 4(c) are confocal laser micrographs showing the results of Experimental Example 7. FIGS. 5A and 5B are confocal laser micrographs showing the results of Experimental Example 10. FIGS. 6(a) and 6(b) are confocal laser micrographs showing the results of Experimental Example 13. FIGS. 7(a) and 7(b) are confocal laser micrographs showing the results of Experimental Example 16. FIG. 8(a) is a schematic diagram showing the structure of DD-YFP used in Experimental Example 18. FIGS. 8(b) and 8(c) are confocal laser micrographs showing the results of Experimental Example 18. FIG. 2 is a schematic diagram illustrating an experimental system for destabilizing mutant eDHFR. FIGS. 10(a) and 10(b) are confocal laser micrographs showing the results of Experimental Example 20. FIG. 11 is a micrograph showing the results of Experimental Example 22. FIG. 12 is a micrograph showing the results of Experimental Example 23. FIG. 13 is a micrograph showing the results of Experimental Example 24. FIG. 14 is a micrograph showing the results of Experimental Examples 26, 29, 30, and 33. FIG. 15 is a micrograph showing the results of Experimental Example 35. FIG. 16(a) is a graph showing the results of calculating the peptide introduction rate of each compound. FIG. 16(b) is a graph showing the results of evaluating the cytotoxicity of each compound. FIGS. 17(a) to (c) are schematic diagrams illustrating inhibition experiments of the PI3K/Akt pathway. FIG. 18(a) is a photograph showing the results of Western blotting in Experimental Example 37. FIG. 18(b) is a graph digitizing the results of FIG. 18(a).

In one embodiment, the present invention provides a compound represented by the following formula (1).

［式（１）中、Ｘ^１は水素原子、アセチル基又は－Ｌ^２－Ｒ^２を表し、Ｘ^２は－ＮＨ_２、－ＯＨ又は－Ｌ^２－Ｒ^２を表し、Ｘ^１とＸ^２の少なくとも一方は－Ｌ^２－Ｒ^２であり、Ｌ^１及びＬ^２は、それぞれ独立に、１又は複数の－Ｃ－が、－Ｏ－、－ＣＯ－、－ＮＨ－、－Ｓ－又は－Ｓ－Ｓ－に置換されていてもよい炭素数１～５０の２価の炭化水素基を表し、Ｒ^１は、イソプレン単位数１～５のプレニル基、炭素数１０～３０の飽和若しくは不飽和のアシル基又は炭素数１０～３０の飽和若しくは不飽和のアルキル基を表し、Ｒ^２は、結合性官能基、又は、分子量１００～５０００の細胞膜非透過性の物質から誘導された１価の基を表し、ｍ、ｎは、それぞれ独立に０～２０の整数を表し、ｐ、ｑは、それぞれ独立に１～４の整数を表す。］

[In formula (1), X ¹ represents a hydrogen atom, an acetyl group, or -L ² -R ² , X ² represents -NH ₂ , -OH or -L ² -R ² , and the combination of X ¹ and X ² At least one is -L ² -R ² , and L ¹ and L ² each independently represent one or more -C-, -O-, -CO-, -NH-, -S- or -S Represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be substituted with -S-, R ¹ is a prenyl group having 1 to 5 isoprene units, or a saturated or unsaturated group having 10 to 30 carbon atoms. It represents an acyl group or a saturated or unsaturated alkyl group having 10 to 30 carbon atoms, and R ² represents a binding functional group or a monovalent group derived from a cell membrane-impermeable substance with a molecular weight of 100 to 5000. where m and n each independently represent an integer of 0 to 20, and p and q each independently represent an integer of 1 to 4. ]

As described later in Examples, when the compound of this embodiment is brought into contact with animal cells, it permeates the cell membrane and is introduced into the cells. In this process, the compound of the present embodiment is introduced into the cytoplasm by endocytosis, or by directly permeating the cell membrane without endocytosis.

Unlike conventional methods, the compound of this embodiment can be introduced into the cytoplasm simply by adding it to the culture medium without pretreating the cells with another compound. Furthermore, by administering the compound of this embodiment to a living body, it can be delivered to the cytoplasm of the living body's cells by endocytosis, or it can be directly permeated through the cell membrane without endocytosis and delivered to the cytoplasm of the living body's cells. can be delivered.

In the compound represented by formula (1), R ² may be a binding functional group or a monovalent group derived from a cell membrane-impermeable substance having a molecular weight of 100 to 5,000.

Examples of the binding functional group include an azide group, a dibenzylcyclooctyne (DBCO) group, a succinimidyl ester group, a maleimide group, a thiol group, an aldehyde group, and a keto group.

A compound in which R ² is a binding functional group can be bonded to any cell membrane-impermeable substance with a molecular weight of 100 to 5,000 through a binding reaction using the binding functional group, thereby allowing the cell membrane-impermeable substance to permeate the cell membrane. It can be used to convert into sexual substances.

For example, when the binding functional group is a DBCO group, the compound represented by formula (1) can be brought into contact with a compound having an azide group to form a bond through a click reaction. Further, for example, when the binding functional group is a succinimidyl ester group, the compound represented by formula (1) can be brought into contact with a compound having a primary amine to form an amide bond to form the bond. I can do it. Further, for example, when the binding functional group is a maleimide group, a thioether bond can be formed and bonded by bringing the compound represented by formula (1) into contact with a compound having a thiol group.

In the compound of this embodiment, the monovalent group derived from a cell membrane-impermeable substance with a molecular weight of 100 to 5,000 is, for example, a group obtained by removing an azide group from a cell membrane-impermeable substance with a molecular weight of 100 to 5,000. , a group excluding an amino group, a group excluding a thiol group, etc.

A substance that does not permeate cell membranes means, for example, a substance that is not introduced into cells even if added to the culture medium of animal cells. For example, a substance whose concentration in the cytoplasm is less than 5 nM after being added to a medium at a concentration of 5 μM and incubated for 30 minutes can be determined to be a substance that does not permeate the cell membrane.

The cell membrane-impermeable substance with a molecular weight of 100 to 5,000 may be a peptide. The peptide may be a linear peptide or a cyclic peptide. Furthermore, the peptide may be composed only of natural amino acids or may be a peptide containing special amino acids. Examples of peptides containing special amino acids include peptoids. Examples of the special amino acids include, but are not limited to, acetylated amino acids, chloroacetylated amino acids, D-amino acids, N-methyl amino acids, β-amino acids, and the like.

Examples of cell membrane-impermeable substances with a molecular weight of 100 to 5,000 include pharmaceuticals, research substances, and the like. Drugs with a molecular weight of less than 500 are called low molecule drugs. Furthermore, drugs with a molecular weight of about 500 to 2,000 are called middle-molecule drugs. Examples of middle molecule drugs include peptide drugs.

More specific cell membrane-impermeable substances with a molecular weight of 100 to 5,000 include, but are not limited to, HA peptide, SH2 peptide, GCN4 peptide, PI3K inhibitor peptide, fluorescein, etc., which will be described later in the Examples.

［式（２）及び式（２’）中、Ｌ^１及びＬ^２は、それぞれ独立に、１又は複数の－Ｃ－が、－Ｏ－、－ＣＯ－、－ＮＨ－、－Ｓ－又は－Ｓ－Ｓ－に置換されていてもよい炭素数１～５０の２価の炭化水素基を表し、Ｒ^１は、イソプレン単位数１～５のプレニル基、炭素数１０～３０の飽和若しくは不飽和のアシル基又は炭素数１０～３０の飽和若しくは不飽和のアルキル基を表し、ｍ、ｎは、それぞれ独立に０～２０の整数を表し、ｐ、ｑは、それぞれ独立に１～４の整数を表す。また、式（２）中、Ｘ^３は、－ＮＨ_２又は－ＯＨを表し、式（２’）中、Ｘ^４は、水素原子又はアセチル基を表す。］ In one embodiment, the present invention provides a method for modifying a cell membrane-impermeable substance with a molecular weight of 100 to 5,000 into a cell-permeable substance, wherein the cell membrane-impermeable substance is represented by the following formula (2). or a group represented by the following formula (2') is provided.

[In formula (2) and formula (2'), L ¹ and L ² each independently represent one or more -C-, -O-, -CO-, -NH-, -S- or - Represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be substituted with SS-, R ¹ is a prenyl group having 1 to 5 isoprene units, or a saturated or unsaturated group having 10 to 30 carbon atoms. represents an acyl group or a saturated or unsaturated alkyl group having 10 to 30 carbon atoms, m and n each independently represent an integer of 0 to 20, and p and q each independently represent an integer of 1 to 4. represent. Further, in formula (2), X ³ represents -NH ₂ or -OH, and in formula (2'), X ⁴ represents a hydrogen atom or an acetyl group. ]

For example, a compound represented by the above formula (1) can be obtained by bonding a group represented by the above formula (2) or a group represented by the above formula (2') to a drug that does not have cell membrane permeability. be able to. As a result, drugs that do not have cell membrane permeability can be introduced into the cytoplasm by endocytosis, or drugs that can be introduced into the cytoplasm by directly permeating the cell membrane without endocytosis, and exhibit their drug efficacy. can be converted to . Thereby, for example, peptide drug discovery can be dramatically accelerated.

Examples of the method for bonding the group represented by the above formula (2) or the group represented by the above formula (2') to a substance that does not have cell membrane permeability include the methods described above. Specifically, in the compound represented by formula (1), R ² is a binding functional group, and by contacting the compound with a substance that does not have cell membrane permeability, the binding functional group and cell membrane permeability are separated. The group represented by the above formula (2) or the group represented by the above formula (2') can be bonded to a substance that does not have cell membrane permeability through a bonding reaction with a substance that does not have cell membrane permeability.

When a cell membrane-impermeable substance with a molecular weight of 100 to 5,000 is a drug, the compound represented by formula (1) above can be said to be a drug delivery system that directly delivers the drug to the cytoplasm.

The cell membrane-impermeable substance with a molecular weight of 100 to 5000 may be a research substance. Research substances are not particularly limited, and include, for example, compound libraries, labeling substances, sugars, and the like. The sugar may be an oligosaccharide. Examples of compound libraries include natural compound libraries, synthetic compound libraries, existing drug libraries, and peptide libraries.

In the compound represented by the above formula (1), the group represented by the above formula (2), or the group represented by the above formula (2'), L ¹ is such that one or more -C- is -O Represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be substituted with -, -CO-, -NH-, -S- or -SS-. L ¹ is not particularly limited as long as it is a group that functions as a linker that connects the group represented by R ¹ and the group represented by the following formula (3). Further, the length of ^L1 is not particularly limited as long as the effects of the present invention can be obtained. L ¹ may be a group derived from the side chain of an amino acid.

［式（３）中、Ｘ^１は水素原子、アセチル基又は－Ｌ^２－Ｒ^２を表し、Ｘ^２は－ＮＨ_２、－ＯＨ又は－Ｌ^２－Ｒ^２を表し、Ｘ^１とＸ^２の少なくとも一方は－Ｌ^２－Ｒ^２であり、Ｌ^２は、それぞれ独立に、１又は複数の－Ｃ－が、－Ｏ－、－ＣＯ－、－ＮＨ－、－Ｓ－又は－Ｓ－Ｓ－に置換されていてもよい炭素数１～５０の２価の炭化水素基を表し、Ｒ^２は、結合性官能基、又は、分子量１００～５０００の細胞膜非透過性の物質から誘導された１価の基を表し、ｍ、ｎは、それぞれ独立に０～２０の整数を表し、ｐ、ｑは、それぞれ独立に１～４の整数を表す。］

[In formula (3), X ¹ represents a hydrogen atom, an acetyl group, or -L ² -R ² , X ² represents -NH ₂ , -OH or -L ² -R ² , and the combination of X ¹ and X ² At least one of them is -L ² -R ² , and each L ² independently represents one or more -C-, -O-, -CO-, -NH-, -S- or -S-S- represents a divalent hydrocarbon group having ¹ to 50 carbon atoms which may be substituted with m and n each independently represent an integer of 0 to 20, and p and q each independently represent an integer of 1 to 4. ]

In the compound represented by the above formula (1), the group represented by the above formula (2), or the group represented by the above formula (2'), L ² is a group represented by R ² and the following formula The group is not particularly limited as long as it functions as a linker for bonding the group represented by (4) or the group represented by the following formula (4'). Further, the length of ^L2 is not particularly limited as long as the effect of the present invention can be obtained. Moreover, in the compound represented by the above formula (1), L ² may include a part of the above-mentioned binding functional group.

［式（４）及び式（４’）中、Ｌ^１は、１又は複数の－Ｃ－が、－Ｏ－、－ＣＯ－、－ＮＨ－、－Ｓ－又は－Ｓ－Ｓ－に置換されていてもよい炭素数１～５０の２価の炭化水素基を表し、Ｒ^１は、イソプレン単位数１～５のプレニル基、炭素数１０～３０の飽和若しくは不飽和のアシル基又は炭素数１０～３０の飽和若しくは不飽和のアルキル基を表し、ｍ、ｎは、それぞれ独立に０～２０の整数を表し、ｐ、ｑは、それぞれ独立に１～４の整数を表す。また、式（４）中、Ｘ^３は、－ＮＨ_２又は－ＯＨを表し、式（４’）中、Ｘ^４は、水素原子又はアセチル基を表す。］

[In formula (4) and formula (4'), L ¹ is one or more -C- is substituted with -O-, -CO-, -NH-, -S- or -S-S- R 1 represents a divalent hydrocarbon group having 1 to 50 carbon atoms, which may have 1 to 5 carbon atoms, and R ¹ is a prenyl group having 1 to 5 isoprene units, a saturated or unsaturated acyl group having 10 to 30 carbon atoms, or a C 10 ~30 saturated or unsaturated alkyl groups, m and n each independently represent an integer of 0 to 20, and p and q each independently represent an integer of 1 to 4. Furthermore, in formula (4), X ³ represents -NH ₂ or -OH, and in formula (4'), X ⁴ represents a hydrogen atom or an acetyl group. ]

As described later in the Examples, after introducing the compound represented by the above formula (1) into the cytoplasm, it is desired to cleave the group represented by R ² and the group represented by the above formula (4). In this case, for example, L ² may contain an -SS- group. In this case, the compound represented by the above formula (1) is introduced into the cytoplasm and then cleaved under reducing conditions within the cytoplasm.

In the compound represented by the above formula (1), the group represented by the above formula (2), or the group represented by the above formula (2'), the compound represented by the following formula (5) or the following formula (6) The group can include a positively charged amino acid residue or a derivative thereof. Examples of positively charged amino acid residues include lysine residues and arginine residues. The group represented by the following formula (5) or the following formula (6) may contain one type of these amino acid residues alone, or may contain a mixture of multiple types. Among others, the group represented by the following formula (5) or the following formula (6) is preferably a lysine residue. As described later in Examples, the use of lysine residues tends to significantly increase the efficiency of introduction into cells while suppressing cytotoxicity.

［式（５）中、ｍ、ｐの定義は上記式（１）におけるものと同様である。］

[In formula (5), the definitions of m and p are the same as those in formula (1) above. ]

［式（６）中、ｎ、ｑの定義は上記式（１）におけるものと同様である。］

[In formula (6), the definitions of n and q are the same as those in formula (1) above. ]

When m in the above formula (5) is 0, the above formula (5) represents a single bond. Similarly, when n in the above formula (6) is 0, the above formula (6) represents a single bond.

When p in the above formula (5) is 4, the group represented by the above formula (5) is a lysine residue of an amino acid or a polymer thereof. Further, when q in the above formula (6) is 4, the group represented by the above formula (6) is a lysine residue of an amino acid or a polymer thereof. The lysine residue may be in the L-configuration or the D-configuration. Further, it may contain both L-form and D-form. Examples of derivatives of lysine residues include groups in which p in the above formula (5) or q in the above formula (6) is other than 4.

When the group represented by the above formula (5) or the above formula (6) contains an L-form lysine residue, after the compound represented by the above formula (1) is introduced into the cytoplasm, the above formula (5) ) or the group represented by the above formula (6) tends to be easily cleaved. In addition, when the group represented by the above formula (5) or the above formula (6) contains a D-type lysine residue, after the compound represented by the above formula (1) is introduced into the cytoplasm, the above formula (5) or the group represented by the above formula (6) tends to be difficult to cleave.

In the group represented by the above formula (5), when m is 2 or more, each repeating unit may be the same or different. Similarly, in the group represented by the above formula (6), when n is 2 or more, each repeating unit may be the same or different.

As will be described later in Examples, by appropriately adjusting the number of m or n, the efficiency of introduction of the compound represented by formula (1) into cells, cytotoxicity, etc. can be adjusted.

For example, the sum of m and n in the above formula (1) may be 5 or more. As described later in Examples, the larger the sum of m and n in the above formula (1), the higher the efficiency of introducing the compound represented by the above formula (1) into cells tends to be. For example, n in the above formula (1) may be 0, and m may be 5 or more.

Moreover, the sum of m and n in the above formula (1) may be 10 or less. As described later in Examples, when the sum of m and n is 10 or less, the cytotoxicity of the compound represented by the above formula (1) tends to be reduced. For example, n in the above formula (1) may be 0, and m may be 10 or less.

Further, in the compound represented by the above formula (1), R ¹ is preferably close to an end of the compound. Further, it is preferable that R ¹ is separated from R ² . More specifically, when X ¹ in the above formula (1) is -L ² -R ² , n is preferably small and m is preferably large. Further, when X ² in the above formula (1) is -L ² -R ² , n is preferably large and m is preferably small.

For example, when X ¹ in the above formula (1) is -L ² -R ² , n in the above formula (1) is preferably 7 or less, preferably 6 or less, and n is 5 It is preferably the following, n is preferably 4 or less, n is preferably 3 or less, n is preferably 2 or less, n is preferably 1 or less, n is 0 It is preferable that

Further, when X ² in the above formula (1) is -L ² -R ² , m in the above formula (1) is preferably 7 or less, m is preferably 6 or less, and m is 5 or less. preferably below, m is preferably 4 or below, m is preferably 3 or below, m is preferably 2 or below, m is preferably 1 or below, m is 0 It is preferable that

When m and n in the above formula (1) are within the above ranges, the efficiency of introducing the compound represented by the above formula (1) into cells tends to increase.

In the compound represented by the above formula (1) or (2), R ¹ is a prenyl group having 1 to 5 isoprene units, a saturated or unsaturated acyl group having 10 to 30 carbon atoms, or a saturated or unsaturated acyl group having 10 to 30 carbon atoms. Represents a saturated or unsaturated alkyl group. More specific examples of R ¹ include, but are not limited to, farnesyl groups, palmitoyl groups, oleoyl groups, etc., which will be described later in Examples.

As will be described later in the Examples, because R ¹ has such a structure, the compound represented by the above formula (1) can be converted into a compound represented by the above formula (1) simply by contacting the compound represented by the above formula (1) with animal cells. It can be introduced into the cytoplasm by endocytosis, or by directly permeating the cell membrane without endocytosis.

Further, after the compound represented by the above formula (1) is introduced into the cytoplasm, when L ² in the above formula (1) or the formula (5) in the above formula (1) is cleaved, ^R2 will be localized within the cytoplasm.

On the other hand, when L ² in the above formula (1) or the formula (5) in the above formula (1) is not cleaved after the compound represented by the above formula (1) is introduced into the cytoplasm. , ^R2 will be localized to specific regions of the cell depending on the nature of ^R1 . Specific regions include, for example, cell membranes.

As described later in the Examples, the compound represented by the above formula (1) can be synthesized by a method using a peptide solid phase synthesis method.

Specific compounds represented by formula (1) include, but are not limited to, compounds (7) to (19), (26), etc., which will be described later in Examples.

In the compound represented by formula (1), when R ² is a group derived from a pharmaceutical, the compound represented by formula (1) can be said to be a pharmaceutical compound. The pharmaceutical compound may be mixed with a pharmaceutically acceptable carrier and formulated as a pharmaceutical composition.

Examples of formulations in pharmaceutical compositions are not particularly limited, and include those used orally as tablets, capsules, and elixirs. Alternatively, examples include those used parenterally in the form of injections of sterile solutions or suspensions mixed with water or other pharmaceutically acceptable liquids.

Examples of additives that can be mixed into tablets and capsules include binders such as gelatin, corn starch, gum tragacanth, and gum arabic, excipients such as crystalline cellulose, leavening agents such as corn starch, gelatin, and alginic acid, and stearin. Examples include lubricants such as magnesium acid, sweeteners such as sucrose, lactose, and saccharin, and flavoring agents such as peppermint and red pepper oil.

Sterile compositions for injection can be formulated according to conventional pharmaceutical practice using a vehicle such as distilled water for injection. Aqueous solutions for injection include, for example, physiological saline, isotonic solutions containing dextrose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizing agents, such as It may be used in combination with alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-50.

It may also be combined with buffers such as phosphate buffers, sodium acetate buffers, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol, and antioxidants. The prepared injection solution is usually filled into suitable ampoules.

Administration to the patient is carried out by, for example, intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular, percutaneous, or oral methods known to those skilled in the art. sell. Although the dosage varies depending on the patient's weight, age, administration method, etc., those skilled in the art can appropriately select an appropriate dosage.

The dosage of the compound varies depending on the drug to be administered and the patient's symptoms, but in the case of oral administration, it is generally about 0.1 to 100 mg per day for an adult (assuming a body weight of 60 kg), for example about 1 .0 to 50 mg, such as about 1.0 to 20 mg.

When administered parenterally, the single dose varies depending on the drug to be administered, the subject to be administered, the target organ, the patient's symptoms, and the administration method, but for example, in the form of an injection, it is usually administered to an adult (assuming a body weight of 60 kg). ), it is generally considered appropriate to administer about 0.01 to 30 mg, for example about 0.1 to 20 mg, for example about 0.1 to 10 mg, by intravenous injection per day.

In one embodiment, the present invention provides a method for introducing a cell membrane-impermeable substance with a molecular weight of 100 to 5,000 into the cytoplasm of a target cell, the method comprising a group represented by the above formula (2) or the above formula (2'). The present invention provides a method comprising the step of contacting the target cell with the substance bound to the target cell.

The method of this embodiment may be performed in vitro or in vivo. Furthermore, the target cells may be cells outside the body or cells within the body of a human or non-human animal.

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[Experiment example 1]
(Synthesis of DBCO- ^D KnFar (n=4,6,8,10))
A compound represented by the following formula (7) (hereinafter sometimes referred to as "DBCO- ^D KnFar (n=4,6,8,10)") was synthesized according to the following scheme (1).

Specifically, as shown in Scheme (1), DBCO- ^D Kn ^D C (n=4, 6, 8, 10) was synthesized.

Fmoc deprotection was performed using 20% piperidine in N,N-dimethylformamide (DMF) for 15 minutes at room temperature. The amino acid coupling reaction was performed using Fmoc-protected amino acids (3.1 equivalents), 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU, 3.0 (eq.), 1-hydroxybenzotriazole (HOBt, 3.0 eq.) and N,N-diisopropylethylamine (DIPEA, 6.0 eq.) at room temperature. All Fmoc deprotection and coupling steps were also monitored by Kaiser test. Also, all washing procedures were performed using DMF.

First, Sieber amide resin (0.79 mmol/g) (127 mg, 100 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc deprotection and coupling reactions were repeated using Fmoc-D-Cys(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Adox-OH, and DBCO aicd as building blocks.

Trt, Boc deprotection and cleavage from the resin was performed using trifluoroacetic acid (TFA) containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and purified with DBCO. - Obtained ^{D K8} DC.

Subsequently, DBCO- ^D Kn ^D C (n = 4, 6, 8, 10), zinc acetate dihydrate, Trans, trans-farnesyl bromide was dissolved and farnesylated. Subsequently, it was purified by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and purified by DBCO- ^D KnFar (n=4,6, 8,10) was obtained as a transparent solid.

MALDI-TOF-MS:
DBCO- ^D K4Far, calcd for [M+H] ⁺ , 1559.9270; found, 1560.1452
DBCO- ^D K6Far, calcd for [M+H] ⁺ , 1816.1169; found, 1817.2498
DBCO- ^D K8Far, calcd for [M+H] ⁺ , 2072.3068; found, 2073.3882
DBCO- ^D K10Far, calcd for [M+H] ⁺ , 2328.4967; found, 2329.5068

[Experiment example 2]
(Synthesis of SH2peptide- ^D K8Far)
A compound represented by the following formula (8) (hereinafter sometimes referred to as "SH2peptide- ^D K8Far") was synthesized according to the following scheme (2).

Specifically, first, Rink amide resin (0.43 mmol/g) (46.5 mg, 20 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc-Lys(N ₂ )-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Pro-OH, Fmoc-Val-OH, Fmoc-F ₂ Pmp-OH, Fmoc-Asp(OtBu )-OH and acetic anhydride as building blocks, Fmoc deprotection and coupling reactions were repeated.

tBu deprotection and cleavage from the resin was performed using TFA containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and purified with SH2peptide. (SH2 peptide) was obtained. The amino acid sequence of SH2peptide was DXVPMLK (SEQ ID NO: 1, X=phosphonodifluoromethylphenylalanine (F2Pmp)), and the molecular weight was about 1045.09.

Subsequently, DBCO- ^D K8Far and SH2 peptide were dissolved in a mixed solution of water/acetonitrile = 1/1 and subjected to a click reaction to link DBCO- ^D K8Far and SH2 peptide. Subsequent purification by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA yielded SH2peptide- ^D K8Far as a white solid.

MALDI-TOF-MS: calcd for [MH] ^- , 3114.7112; found, 3115.7249

[Experiment example 3]
(Study of localization using SH2peptide- ^D K8Far)
HeLa cells expressing Src homology 2 domain (SH2 domain)-EGFP (hereinafter sometimes referred to as "SH2-EGFP") derived from the C-terminus of phosphatidylinositol 3-kinase (PI3K) (hereinafter referred to as "SH2-EGFP expression") SH2 peptide- ^D K8Far was added to a final concentration of 10 μM to the culture medium of the cells. Subsequently, EGFP fluorescence was observed using a confocal laser microscope.

FIG. 1(a) is a schematic diagram showing the structure of SH2-EGFP. The amino acid sequence of SH2-EGFP is shown in SEQ ID NO:2. FIGS. 1(b) and 1(c) are photographs showing the results of observation using a confocal laser microscope. Scale bar is 10 μm.

FIG. 1(b) is a photograph of SH2-EGFP-expressing cells before addition of SH2peptide- ^D K8Far. Furthermore, FIG. 1(c) is a photograph of SH2-EGFP-expressing cells taken 30 minutes after the addition of SH2peptide- ^D K8Far. As a result, it was revealed that SH2-EGFP was localized to the cell membrane 30 minutes after the addition of SH2peptide- ^D K8Far.

This result suggests that SH2peptide ^-D K8Far penetrates the cell membrane of SH2-EGFP-expressing cells and is introduced into the cytoplasm, and the SH2 domain of SH2-EGFP present in the cytoplasm binds to the SH2peptide of SH2peptide ^-D K8Far to form a complex. Furthermore, it is shown that the formed complex was localized to the cell membrane due to the presence of the farnesyl group of SH2peptide- ^D K8Far.

FIG. 2 is a schematic diagram illustrating an experimental system for cell membrane localization. The "carrier molecule" in FIG. 2 corresponds to SH2peptide- ^D K8Far in this experimental example. “Target Protein-Fluorescent Protein” in FIG. 2 corresponds to SH2-EGFP in this experimental example. As shown in FIG. 2, when SH2peptide- ^D K8Far is brought into contact with SH2-EGFP-expressing cells, SH2peptide ^-D K8Far passes through the cell membrane and is introduced into the cytoplasm. Subsequently, the SH2 domain of SH2-EGFP present in the cytoplasm binds to the SH2 peptide of SH2 peptide- ^D K8Far to form a complex. Subsequently, the formed complex is localized to the cell membrane due to the presence of the farnesyl group of SH2peptide- ^D K8Far. As a result, EGFP fluorescence is observed in the cell membrane.

[Experiment example 4]
(Synthesis of GCN4peptide- ^D K8Far)
A compound represented by the following formula (9) (hereinafter sometimes referred to as "GCN4peptide- ^D K8Far") was synthesized according to the following scheme (3).

Specifically, first, Rink amide resin (0.43 mmol/g) (69.8 mg, 30 μmol) was deprotected with Fmoc and washed. Subsequently, Fmoc-Lys(N ₂ )-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)- Fmoc deprotection and cupping using OH, Fmoc-His(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, and acetic anhydride as building blocks. The ring reaction was repeated.

Boc, Pbf, OtBu, Trt, tBu deprotection and cleavage from the resin was performed using TFA containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and purified with GCN4 peptide. (GCN4 peptide) was obtained. The amino acid sequence of GCN4 peptide was EELLSKNYHLENEVARLKKK (SEQ ID NO: 3), and the molecular weight was about 2508.87.

Subsequently, DBCO- ^D K8Far and GCN4 peptide were dissolved in a mixed solution of water/acetonitrile = 1/1 and subjected to a click reaction to link DBCO- ^D K8Far and GCN4 peptide. Subsequent purification by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA yielded GCN4peptide ^-D K8Far as a white solid.

MALDI-TOF-MS: calcd for [M+H] ⁺ , 4579.6677; found, 4580.1319

[Experiment example 5]
(Study of localization using GCN4peptide- ^D K8Far)
Single-chain variable fragment GCN4-sfGFP (hereinafter sometimes referred to as "scFv GCN4-sfGFP") was expressed in HeLa cells (hereinafter sometimes referred to as "scFv GCN4-sfGFP expressing cells"). GCN4 peptide- ^D K8Far was added to a concentration of 15 μM. Subsequently, the fluorescence of sfGFP was observed using a confocal laser microscope.

FIG. 3(a) is a schematic diagram showing the structure of scFv GCN4-sfGFP. The amino acid sequence of scFv GCN4-sfGFP is shown in SEQ ID NO: 4. scFv GCN4 is a single chain antibody that binds to the GCN4 peptide. FIGS. 3(b) and 3(c) are photographs showing the results of observation using a confocal laser microscope. Scale bar is 10 μm.

FIG. 3(b) is a photograph of scFv GCN4-sfGFP-expressing cells before addition of GCN4peptide- ^D K8Far. Furthermore, FIG. 3(c) is a photograph of scFv GCN4-sfGFP-expressing cells taken 30 minutes after the addition of GCN4peptide- ^D K8Far. The results revealed that sfGFP was localized to the cell membrane 30 minutes after the addition of GCN4peptide ^-D K8Fa.

This result indicates that GCN4peptide- ^D K8Far penetrates the cell membrane of scFv GCN4-sfGFP expressing cells and is introduced into the cytoplasm, and the scFv GCN4 of scFv GCN4-sfGFP present in the cytoplasm binds to the GCN4 peptide of GCN4peptide ^-D K8Far. Furthermore, the formed complex was localized to the cell membrane due to the presence of the farnesyl group of GCN4peptide ^- DK8Far.

[Experiment example 6]
(Synthesis of HApeptide- ^D K8Far)
A compound represented by the following formula (10) (hereinafter sometimes referred to as "HApeptide- ^D K8Far") was synthesized according to the following scheme (4).

Specifically, first, Rink amide resin (0.43 mmol/g) (69.8 mg, 30 μmol) was deprotected with Fmoc and washed. Subsequently, Fmoc-Lys(N ₂ )-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Val-OH, anhydrous Fmoc deprotection and coupling reactions were repeated using acetic acid as a building block.

OtBu, tBu deprotection and cleavage from the resin was performed using TFA containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse-phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and HApeptide. (HA peptide) was obtained. The amino acid sequence of HApeptide was YPYDVPDPYAK (SEQ ID NO: 5), and the molecular weight was approximately 1297.39.

Subsequently, DBCO- ^D K8Far and HApeptide were dissolved in a mixed solution of water/acetonitrile = 1/1 and a click reaction was performed to link DBCO- ^D K8Far and HApeptide. Subsequent purification by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA yielded HApeptide- ^D K8Far as a white solid.

MALDI-TOF-MS: calcd for [M+H] ⁺ , 3368.8843; found, 3368.3081

[Experiment Example 7]
(Study of localization using HApeptide- ^D K8Far)
A final concentration of 2.5 μM was added to the culture medium of HeLa cells expressing Frankenbody (15F11)-mEGFP (hereinafter sometimes referred to as "15F11-mEGFP") (hereinafter sometimes referred to as "15F11-mEGFP expressing cells"). HApeptide- ^D K8Far was added so that Subsequently, mEGFP fluorescence was observed using a confocal laser microscope.

FIG. 4(a) is a schematic diagram showing the structure of 15F11-mEGFP. The amino acid sequence of 15F11-mEGFP is shown in SEQ ID NO: 6. Frankenbodies are single chain antibodies that bind to HA peptides. FIGS. 4(b) and 4(c) are photographs showing the results of observation using a confocal laser microscope. Scale bar is 10 μm.

FIG. 4(b) is a photograph of 15F11-mEGFP-expressing cells before addition of HApeptide- ^D K8Far. Furthermore, FIG. 4(c) is a photograph of 15F11-mEGFP-expressing cells taken 30 minutes after the addition of HApeptide- ^D K8Far. The results revealed that mEGFP was localized to the cell membrane 30 minutes after the addition of HApeptide- ^D K8Far.

This result indicates that HApeptide- ^D K8Far penetrates the cell membrane of 15F11-mEGFP-expressing cells and is introduced into the cytoplasm, and the Frankenbody (15F11) of 15F11-mEGFP present in the cytoplasm binds to the HA peptide of HApeptide- ^D K8Far and forms a complex. Furthermore, the formed complex was localized to the cell membrane due to the presence of the farnesyl group of HApeptide- ^D K8Far.

[Experiment example 8]
(Synthesis of DBCO- ^D K4Far ^D K4)
A compound represented by the following formula (11) (hereinafter sometimes referred to as "DBCO- ^D K4Far ^D K4") was synthesized according to the following scheme (5).

Specifically, as shown in Scheme (5), DBCO- ^D K4 ^{D CD} K4 was synthesized.

Fmoc deprotection was performed using 20% piperidine in N,N-dimethylformamide (DMF) for 15 minutes at room temperature. The amino acid coupling reaction was performed using Fmoc-protected amino acids (3.1 equivalents), 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU, 3.0 (eq.), 1-hydroxybenzotriazole (HOBt, 3.0 eq.) and N,N-diisopropylethylamine (DIPEA, 6.0 eq.) at room temperature. All Fmoc deprotection and coupling steps were also monitored by Kaiser test. Also, all washing procedures were performed using DMF.

First, Sieber amide resin (0.79 mmol/g) (38 mg, 30 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc deprotection and coupling reactions were repeated using Fmoc-D-Cys(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Adox-OH, and DBCO aicd as building blocks.

Trt, Boc deprotection and cleavage from the resin was performed using trifluoroacetic acid (TFA) containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and purified with DBCO. - ^D K4 ^{D CD} K4 was obtained.

Subsequently, DBCO ^{- DK4} , zinc acetate dihydrate, and ^trans ,trans-farnesyl bromide were dissolved in a mixed solution of DMF/acetonitrile/water = 2/1/1 (0.05% TFA). and farnesylated. Subsequent purification by reverse phase ^HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA yielded DBCO ^{- D} K4 as a transparent solid. obtained as.

MALDI-TOF-MS: calcd for [M+H] ⁺ , 1868.1190; found, 1868.2736

[Experiment example 9]
(Synthesis of HApeptide- ^D K4Far ^D K4)
A compound represented by the following formula (12) (hereinafter sometimes referred to as "HApeptide- ^D K4Far ^D K4") was synthesized according to the following scheme (6).

Subsequently, DBCO- ^D K4Far ^D K4 and HApeptide were dissolved in a mixed solution of water/acetonitrile = 1/1 and a click reaction was performed to link DBCO- ^D K4Far ^D K4 and HApeptide. Subsequent purification by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA yielded HApeptide- ^D K4Far ^D K4 as a white solid. Ta.

MALDI-TOF-MS: calcd for [M+H] ⁺ , 3368.8843; found, 3368.8310

[Experiment example 10]
(Study of localization by HApeptide- ^D K4Far ^D K4)
Frankenbody (15F11)-mEGFP (hereinafter sometimes referred to as "15F11-mEGFP") is expressed in the culture medium of HeLa cells (hereinafter sometimes referred to as "15F11-mEGFP expressing cells") at a final concentration of 10 μM. HApeptide- ^D K4Far ^D K4 was added as follows. Subsequently, mEGFP fluorescence was observed using a confocal laser microscope.

FIGS. 5(a) and 5(b) are photographs showing the results of observation using a confocal laser microscope. Scale bar is 10 μm.

FIG. 5(a) is a photograph of 15F11-mEGFP-expressing cells before addition of HApeptide- ^D K4Far ^D K4. Furthermore, FIG. 5(b) is a photograph of 15F11-mEGFP-expressing cells taken 30 minutes after the addition of HApeptide ^-D K4Far ^D K4. The results revealed that mEGFP was localized to the cell membrane 30 minutes after the addition of HApeptide- ^D K4Far ^D K4.

This result indicates that HApeptide- ^D K4Far ^D K4 penetrates the cell membrane of 15F11-mEGFP-expressing cells and is introduced into the cytoplasm, and the Frankenbody (15F11) of 15F11-mEGFP present in the cytoplasm interacts with the HA peptide of HApeptide ^-D K4Far ^D K4. It is shown that HApeptide-D K4 binds to form a complex, and that the formed complex is localized in the cell membrane due to the presence of the farnesyl group of HApeptide ^-D K4Far ^D K4.

This result shows that even if R ¹ (farnesyl group in this experimental example) in the above formula (1) is not located at the terminal of the compound represented by the above formula (1), This shows that the compound treated can permeate the cell membrane.

[Experiment example 11]
(Synthesis of DBCO- ^D K8Pal)
A compound represented by the following formula (13) (hereinafter sometimes referred to as "DBCO- ^D K8Pal") was synthesized according to the following scheme (7).

Specifically, as shown in Scheme (7), DBCO- ^D K8Pal was synthesized.

Fmoc deprotection was performed using 20% piperidine in N,N-dimethylformamide (DMF) for 15 minutes at room temperature. ivDde deprotection was performed using 5% hydrazine in N,N-dimethylformamide (DMF) for 30 minutes at room temperature. The amino acid coupling reaction was performed using Fmoc-protected amino acids (3.1 equivalents), 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU, 3.0 (eq.), 1-hydroxybenzotriazole (HOBt, 3.0 eq.) and N,N-diisopropylethylamine (DIPEA, 6.0 eq.) at room temperature. All Fmoc deprotection and coupling steps were also monitored by Kaiser test. Also, all washing procedures were performed using DMF.

First, Sieber amide resin (0.79 mmol/g) (38 mg, 30 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc deprotection and coupling reactions were repeated using Fmoc-D-Lys(ivDde)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Adox-OH, and DBCO aicd as building blocks. Finally, ivDde deprotection was performed, and palmitic acid was coupled.

Boc deprotection and cleavage from the resin was performed using trifluoroacetic acid (TFA) containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and purified with DBCO. - ^D K8Pal was obtained.

MALDI-TOF-MS: calcd for [M+H] ⁺ , 2040.4359; found, 2040.5491

[Experiment example 12]
(Synthesis of HApeptide- ^D K8Pal)
A compound represented by the following formula (14) (hereinafter sometimes referred to as "HApeptide- ^D K8Pal") was synthesized according to the following scheme (8).

Subsequently, DBCO- ^D K8Pal and HApeptide were dissolved in a mixed solution of water/acetonitrile = 1/1 and a click reaction was performed to link DBCO- ^D K8Pal and HApeptide. Subsequent purification by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA yielded HApeptide- ^D K8Pal as a white solid.

MALDI-TOF-MS: calcd for [M+H] ⁺ , 3598.1175; found, 3599.6700

[Experiment example 13]
(Study of localization using HApeptide- ^D K8Pal)
A final concentration of 2.5 μM was added to the medium of HeLa cells expressing Frankenbody (15F11)-mEGFP (hereinafter sometimes referred to as "15F11-mEGFP") (hereinafter sometimes referred to as "15F11-mEGFP expressing cells"). HApeptide- ^D K8Pal was added so that Subsequently, mEGFP fluorescence was observed using a confocal laser microscope.

FIGS. 6(a) and 6(b) are photographs showing the results of observation using a confocal laser microscope. Scale bar is 10 μm.

FIG. 6(a) is a photograph of 15F11-mEGFP-expressing cells before addition of HApeptide- ^D K8Pal. Furthermore, FIG. 6(b) is a photograph of 15F11-mEGFP-expressing cells taken 30 minutes after the addition of HApeptide- ^D K8Pal. The results revealed that mEGFP was localized to the cell membrane 30 minutes after the addition of HApeptide- ^D K8Pal.

This result indicates that HApeptide- ^D K8Pal permeates the cell membrane of 15F11-mEGFP-expressing cells and is introduced into the cytoplasm, and the Frankenbody (15F11) of 15F11-mEGFP present in the cytoplasm binds to the HA peptide of HApeptide- ^D K8Pal to form a complex. Furthermore, the formed complex was localized to the cell membrane due to the presence of the palmitoyl group of HApeptide- ^D K8Pal.

This result shows that the compound represented by the above formula (1) can permeate the cell membrane even if R ¹ in the above formula (1) is a group other than a farnesyl group.

[Experiment example 14]
(Synthesis of DBCO- ^D K8Ole)
A compound represented by the following formula (15) (hereinafter sometimes referred to as "DBCO- ^D K8Ole") was synthesized according to the following scheme (9).

Specifically, as shown in Scheme (9), DBCO- ^D K8Pal was synthesized.

Fmoc deprotection was performed using 20% piperidine in N,N-dimethylformamide (DMF) for 15 minutes at room temperature. ivDde deprotection was performed using 5% hydrazine in N,N-dimethylformamide (DMF) for 30 minutes at room temperature. The amino acid coupling reaction was performed using Fmoc-protected amino acids (3.1 equivalents), 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU, 3.0 (eq.), 1-hydroxybenzotriazole (HOBt, 3.0 eq.) and N,N-diisopropylethylamine (DIPEA, 6.0 eq.) at room temperature. All Fmoc deprotection and coupling steps were also monitored by Kaiser test. Also, all washing procedures were performed using DMF.

First, Sieber amide resin (0.79 mmol/g) (38 mg, 30 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc deprotection and coupling reactions were repeated using Fmoc-D-Lys(ivDde)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Adox-OH, and DBCO aicd as building blocks. Finally, ivDde deprotection was performed and Oleic acid was coupled.

Boc deprotection and cleavage from the resin was performed using trifluoroacetic acid (TFA) containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and purified with DBCO. - ^D K8Ole was obtained.

MALDI-TOF-MS: calcd for [M+H] ⁺ , 2066.4515; found, 2066.4922

[Experiment example 15]
(Synthesis of HApeptide- ^D K8Ole)
A compound represented by the following formula (16) (hereinafter sometimes referred to as "HApeptide- ^D K8Ole") was synthesized according to the following scheme (10).

Subsequently, DBCO- ^D K8Ole and HApeptide were dissolved in a mixed solution of water/acetonitrile = 1/1 and a click reaction was performed to link DBCO ^- DK8Ole and HApeptide. Subsequent purification by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA yielded HApeptide- ^D K8Ole as a white solid.

MALDI-TOF-MS: calcd for [MH] ^- , 3622.1186; found, 3623.2365

[Experiment example 16]
(Study of localization using HApeptide- ^D K8Ole)
A final concentration of 2.5 μM was added to the medium of HeLa cells expressing Frankenbody (15F11)-mEGFP (hereinafter sometimes referred to as "15F11-mEGFP") (hereinafter sometimes referred to as "15F11-mEGFP expressing cells"). HApeptide- ^D K8Ole was added so that Subsequently, mEGFP fluorescence was observed using a confocal laser microscope.

FIGS. 7A and 7B are photographs showing the results of observation using a confocal laser microscope. Scale bar is 10 μm.

FIG. 7(a) is a photograph of 15F11-mEGFP-expressing cells before addition of HApeptide- ^D K8Ole. Furthermore, FIG. 7(b) is a photograph of 15F11-mEGFP-expressing cells taken 30 minutes after the addition of HApeptide- ^D K8Ole. The results revealed that mEGFP was localized to the cell membrane 30 minutes after the addition of HApeptide- ^D K8Ole.

This result indicates that HApeptide- ^D K8Ole is introduced into the cytoplasm through the cell membrane of 15F11-mEGFP-expressing cells, and the Frankenbody (15F11) of 15F11-mEGFP present in the cytoplasm binds to the HA peptide of HApeptide ^-D K8Ole and forms a complex. Furthermore, the formed complex was localized in the cell membrane due to the presence of the oleoyl group of HApeptide- ^D K8Ole.

This result further indicates that the compound represented by the above formula (1) can permeate the cell membrane even if R ¹ in the above formula (1) is a group other than a farnesyl group.

[Experiment example 17]
(Synthesis of HApeptide-TMP-SS- ^D K8Far)
A compound represented by the following formula (17) (hereinafter sometimes referred to as "HApeptide-TMP-SS- ^D K8Far") was synthesized according to the following scheme (11).

Specifically, first, Sieber amide resin (0.79 mmol/g) (38.0 mg, 30 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc-Lys(N ₂ )-OH, Fmoc-NH-ethyl-SS-propionic aicd, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc- Fmoc deprotection and coupling reactions were repeated using Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Val-OH, and acetic anhydride as building blocks. Finally, ivDde deprotection was performed and TMP acid was coupled.

OtBu, tBu deprotection and cleavage from the resin was performed using TFA containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse-phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and HApeptide. I got it. The amino acid sequence of HApeptide was YPYDVPDPYAK (SEQ ID NO: 5), and the molecular weight was approximately 1947.22.

Subsequently, DBCO- ^D K8Far and HApeptide-TMP-SS-N3 were dissolved in a mixed solution of water/acetonitrile = 1/1 and a click reaction was performed to link DBCO- ^D K8Far and HApeptide-TMP-SS-N3. . Subsequent purification by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA yielded HApeptide-TMP-SS- ^D K8Far as a white solid. obtained as.

MALDI-TOF-MS: calcd for [M+H] ⁺ , 4018.1559; found, 4018.1715

[Experiment example 18]
(Study of intracellular introduction of HApeptide-TMP-SS- ^D K8Far)
eDHFR (Destabilized Domain)-YFP (hereinafter sometimes referred to as "DD-YFP") was added to the culture medium of HeLa cells (hereinafter sometimes referred to as "DD-YFP expressing cells") at a final concentration of 1 μM. HApeptide-TMP-SS- ^D K8Far was added so that Subsequently, YFP fluorescence was observed using a confocal laser microscope.

FIG. 8(a) is a schematic diagram showing the structure of DD-YFP. The amino acid sequence of DD-YFP is shown in SEQ ID NO: 7. After DD-YFP is expressed within cells, it is rapidly degraded. On the other hand, when TMP, which is a ligand for eDHFR, binds to DD-YFP, it is stabilized and no longer degraded, thereby increasing the fluorescence intensity of YFP within the cell.

FIGS. 8(b) and 8(c) are photographs showing the results of observation using a confocal laser microscope. Scale bar is 20 μm. FIG. 8(b) is a photograph of DD-YFP expressing cells before addition of HApeptide-TMP-SS- ^D K8Far. Furthermore, FIG. 8(c) is a photograph of DD-YFP expressing cells taken 60 minutes after the addition of HApeptide-TMP-SS- ^D K8Far. The results revealed that YFP fluorescence was detected in the cytoplasm 60 minutes after the addition of HApeptide-TMP-SS- ^D K8Far.

This result indicates that HApeptide-TMP-SS ^-D K8Far penetrates the cell membrane of DD-YFP-expressing cells and is introduced into the cytoplasm, and DD-YFP present in the cytoplasm binds to TMP of HApeptide-TMP-SS- ^D K8Far. This shows that a complex was formed in the cells, and that the formed complex diffused into the cytoplasm. Since the cytoplasm is under reducing conditions, disulfide bonds are cleaved after HApeptide-TMP-SS- ^D K8Far is incorporated into the cytoplasm. As a result, HApeptide-TMP and ^DK8Far are separated. Then, HApeptide-TMP binds to DD-YFP, stabilizes DD-YFP, increases the intracellular expression level, and diffuses into the cytoplasm.

FIG. 9 is a schematic diagram illustrating an experimental system for destabilizing mutant eDHFR. The “carrier molecule” in FIG. 9 corresponds to HApeptide-TMP-SS- ^D K8Far in this experimental example. “Destabilized mutant eDHFR-YFP” in FIG. 9 corresponds to DD-YFP in this experimental example. Since DD-YFP is a destabilizing mutant, it is degraded immediately after expression.

As shown in FIG. 9, when HApeptide-TMP-SS ^-D K8Far is brought into contact with DD-YFP-expressing cells, HApeptide-TMP-SS- ^D K8Far passes through the cell membrane and is introduced into the cytoplasm. Subsequently, DD-YFP present in the cytoplasm binds to TMP of HApeptide-TMP-SS- ^D K8Far to form a complex. As a result, DD-YFP is stabilized, no longer degraded, and its abundance increases.

Furthermore, the disulfide bond of HApeptide-TMP-SS- ^D K8Far is cleaved due to the reducing conditions in the cytoplasm. As a result, HApeptide-TMP and ^DK8Far are separated. As a result, DD-YFP diffuses into the cytoplasm.

As described below, when HApeptide-TMP-Aoc- ^{D K8Far, which does not have the disulfide bond of HApeptide-TMP-SS-D K8Far} , is brought into contact with DD-YFP-expressing cells, HApeptide-TMP-Aoc ^-D K8Far is attached to the cell membrane. is introduced into the cytoplasm. Subsequently, DD-YFP present in the cytoplasm binds to TMP of HApeptide-TMP-Aoc- ^D K8Far to form a complex. Here, HApeptide-TMP-Aoc- ^D K8Far is not cleaved even under reducing conditions in the cytoplasm. As a result, the formed complex is localized at the cell membrane due to the presence of the farnesyl group of HApeptide-TMP-Aoc- ^D K8Far. As a result, YFP fluorescence is observed in the cell membrane.

[Experiment example 19]
(Synthesis of HApeptide-TMP-Aoc- ^D K8Far)
A compound represented by the following formula (18) (hereinafter sometimes referred to as "HApeptide-TMP-Aoc- ^D K8Far") was synthesized according to the following scheme (12).

Specifically, first, Sieber amide resin (0.79 mmol/g) (38.0 mg, 30 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc-Lys(N ₂ )-OH, Fmoc-8-Aoc-OH, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu )-OH, Fmoc-Pro-OH, Fmoc-Val-OH, and acetic anhydride as building blocks, Fmoc deprotection and coupling reactions were repeated. Finally, ivDde deprotection was performed and TMP acid was coupled.

OtBu, tBu deprotection and cleavage from the resin was performed using TFA containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse-phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and HApeptide. I got it. The amino acid sequence of HApeptide was YPYDVPDPYAK (SEQ ID NO: 5), and the molecular weight was approximately 1947.22.

Subsequently, DBCO- ^D K8Far and HApeptide-TMP-Aoc-N3 were dissolved in a mixed solution of water/acetonitrile = 1/1 and a click reaction was performed to link DBCO- ^D K8Far and HApeptide-TMP-Aoc-N3. . HApeptide-TMP-Aoc-D K8Far was then purified by reverse-phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA to yield HApeptide-TMP-Aoc- ^D K8Far as a white solid. obtained as.

MALDI-TOF-MS: calcd for [M+H] ⁺ , 3995.2515; found, 3996.3704

[Experiment example 20]
(Study of intracellular introduction of HApeptide-TMP-Aoc- ^D K8Far)
eDHFR (Destabilized Domain)-YFP (hereinafter sometimes referred to as "DD-YFP") was added to the culture medium of HeLa cells (hereinafter sometimes referred to as "DD-YFP expressing cells") at a final concentration of 1 μM. HApeptide-TMP-Aoc- ^D K8Far was added to the solution. Subsequently, YFP fluorescence was observed using a confocal laser microscope.

FIGS. 10(a) and 10(b) are photographs showing the results of observation using a confocal laser microscope. Scale bar is 20 μm.

FIG. 10(a) is a photograph of DD-YFP expressing cells before addition of HApeptide-TMP-Aoc- ^D K8Far. Furthermore, FIG. 10(b) is a photograph of DD-YFP expressing cells taken 60 minutes after the addition of HApeptide-TMP-Aoc- ^D K8Far. The results revealed that DD-YFP was localized to the cell membrane 60 minutes after the addition of HApeptide-TMP-Aoc- ^D K8Far.

This result indicates that HApeptide-TMP-Aoc- ^D K8Far penetrates the cell membrane of DD-YFP-expressing cells and is introduced into the cytoplasm, and the eDHFR of DD-YFP present in the cytoplasm interacts with TMP of HApeptide-TMP-Aoc ^-D K8Far. It is shown that HApeptide-TMP-Aoc-D K8Far binds to form a complex, and that the formed complex is localized in the cell membrane due to the presence of the farnesyl group of HApeptide-TMP-Aoc ^-D K8Far. Note that HApeptide-TMP-Aoc- ^D K8Far is not cleaved even under reducing conditions in the cytoplasm.

[Experiment example 21]
(Synthesis of HApeptide-FL-SS- ^D KnFar (n=4, 6, 8, 10))
According to the following scheme (13), a compound represented by the following formula (19) (hereinafter sometimes referred to as "HApeptide-FL-SS- ^D KnFar (n = 4, 6, 8, 10)") is synthesized. did.

Specifically, first, Sieber amide resin (0.79 mmol/g) (76.0 mg, 60 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc-Lys(N ₂ )-OH, Fmoc-NH-ethyl-SS-propionic aicd, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc- Fmoc deprotection and coupling reactions were repeated using Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Val-OH, and acetic anhydride as building blocks. Finally, ivDde deprotection was performed and 5(6)-Carboxyfluorescein (FL) was coupled.

OtBu, tBu deprotection and cleavage from the resin was performed using TFA containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse-phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and HApeptide. -FL-SS-N3 was obtained. The amino acid sequence of HApeptide was YPYDVPDPYAK (SEQ ID NO: 5), and the molecular weight was approximately 1947.12.

Subsequently, DBCO- ^D KnFar (n=4, 6, 8, 10) and HApeptide-FL-SS-N3 were dissolved in a mixed solution of water/acetonitrile = 1/1 and click-reacted to form DBCO- ^D KnFar. HApeptide-FL-SS-N3 was ligated. It was subsequently purified by reverse-phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA to obtain HApeptide-FL-SS- ^D KnFar (n= 4, 6, 8, 10) were obtained as white solids.

HApeptide-FL-SS- ^D K4Far, calcd for [M+H] ⁺ , 3054.7; found, 3511.3
HApeptide-FL-SS- ^D K6Far, calcd for [M+H] ⁺ , 3760.8; found, 3766.7
HApeptide-FL-SS- ^D K8Far, calcd for [M+H] ⁺ , 4017.03; found, 4017.8
HApeptide-FL-SS- ^D K10Far, calcd for [M+H] ⁺ , 4273.2; found, 4280.6

[Experiment example 22]
(Study of intracellular introduction of HApeptide-FL-SS- ^D KnFar (n=4, 6, 8, 10))
HApeptide-FL-SS- ^D KnFar (n=4, 6, 8, 10) was added to the HeLa cell culture medium at a final concentration of 5 μM or 10 μM. Subsequently, 30 minutes after the addition of HApeptide-FL-SS- ^D KnFar, the fluorescence of FL was observed using a confocal laser microscope. In addition, dead cells were detected by staining with PI (propidium iodide) and detecting PI fluorescence.

FIG. 11 is a photograph showing the results of microscopic observation. Scale bar is 80 μm. In FIG. 11, "FL" indicates a photograph in which fluorescein fluorescence was observed, "PI" indicates a photograph in which PI fluorescence was observed, and "DIC" indicates a differential interference microscope photograph. The results revealed that FL fluorescence was detected 30 minutes after the addition of HApeptide-FL-SS- ^D KnFar (n=4, 6, 8, 10).

This result indicates that HApeptide-FL-SS- ^D KnFar (n = 4, 6, 8, 10) was introduced into the cytoplasm by directly permeating the cell membrane and diffusing into the cytoplasm, rather than by endocytosis. show. More specifically, after HApeptide-FL-SS- ^D KnFar (n = 4, 6, 8, 10) is introduced into the cytoplasm, the disulfide bond is cleaved, HApeptide-FL and ^D KnFar are separated, and HApeptide- It shows that FL diffused into the cytoplasm.

[Experiment example 23]
(Study of intracellular introduction of HApeptide-FL-SS- ^D K8Far)
HApeptide-FL-SS- ^D K8Far was added to the HeLa cell culture medium at a final concentration of 5 μM. Subsequently, FL fluorescence was observed over time using a confocal laser microscope. In addition, dead cells were detected by staining with PI (propidium iodide) and detecting PI fluorescence.

FIG. 12 is a photograph showing the results of microscopic observation. Scale bar is 40 μm. In FIG. 12, "FL" indicates a photograph in which fluorescein fluorescence was observed, "PI" indicates a photograph in which PI fluorescence was observed, and "DIC" indicates a differential interference microscope photograph. Furthermore, "before" indicates a photograph taken before the addition of HApeptide-FL-SS- ^D K8Far. The results revealed that FL fluorescence in the cytoplasm increased over time after the addition of HApeptide-FL-SS- ^D K8Far.

[Experiment example 24]
(inhibition of endocytosis)
The temperature of HeLa cell culture medium was adjusted to 4°C to inhibit endocytosis. Subsequently, HApeptide-FL-SS- ^D K8Far was added to the medium at a final concentration of 5 μM. Subsequently, 30 minutes later, the fluorescence of FL was observed using a confocal laser microscope. In addition, dead cells were detected by staining with PI (propidium iodide) and detecting PI fluorescence.

FIG. 13 is a photograph showing the results of microscopic observation. Scale bar is 40 μm. In FIG. 13, "FL" indicates a photograph in which fluorescein fluorescence was observed, "PI" indicates a photograph in which PI fluorescence was observed, and "DIC" indicates a differential interference microscope photograph. The results revealed that FL fluorescence was detected in the cytoplasm 30 minutes after the addition of HApeptide-FL-SS- ^D K8Far.

This result indicates that HApeptide-FL-SS- ^D K8Far is not introduced into the cytoplasm by endocytosis. That is, it is shown that HApeptide-FL-SS- ^D K8Far directly permeates the cell membrane and is introduced into the cytoplasm.

[Experiment example 24]
(Synthesis of DBCO- ^D K8)
A compound represented by the following formula (20) (hereinafter sometimes referred to as "DBCO- ^D K8") was synthesized according to the following scheme (14).

Specifically, as shown in Scheme (14), DBCO- ^DK8 was synthesized.

Fmoc deprotection was performed using 20% piperidine in N,N-dimethylformamide (DMF) for 15 minutes at room temperature. The amino acid coupling reaction was performed using Fmoc-protected amino acids (3.1 equivalents), 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU, 3.0 (eq.), 1-hydroxybenzotriazole (HOBt, 3.0 eq.) and N,N-diisopropylethylamine (DIPEA, 6.0 eq.) at room temperature. All Fmoc deprotection and coupling steps were also monitored by Kaiser test. Also, all washing procedures were performed using DMF.

First, Rink amide resin (0.43 mmol/g) (46.5 mg, 20 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc deprotection and coupling reactions were repeated using Fmoc-D-Lys(Boc)-OH, Fmoc-Adox-OH, and DBCO acid as building blocks.

Boc deprotection and cleavage from the resin was performed using trifluoroacetic acid (TFA) containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and purified with DBCO. - Obtained ^D K8.

MALDI-TOF-MS: calcd for [M+H] ⁺ , 1765.1098; found, 1765.0429

[Experiment example 25]
(Synthesis of HApeptide-FL-SS- ^D K8)
A compound represented by the following formula (21) (hereinafter sometimes referred to as "HApeptide-FL-SS- ^D K8") was synthesized according to the following scheme (15).

Specifically, first, Sieber amide resin (0.79 mmol/g) (76.0 mg, 60 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc-Lys(N ₂ )-OH, Fmoc-NH-ethyl-SS-propionic aicd, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc- Fmoc deprotection and coupling reactions were repeated using Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Val-OH, and acetic anhydride as building blocks. Finally, ivDde deprotection was performed and 5(6)-Carboxyfluorescein (FL) was coupled.

OtBu, tBu deprotection and cleavage from the resin was performed using TFA containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse-phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and HApeptide. -FL-SS-N3 was obtained. The amino acid sequence of HApeptide was YPYDVPDPYAK (SEQ ID NO: 5), and the molecular weight was approximately 1947.12.

Subsequently, DBCO- ^D K8 and HApeptide-FL-SS-N3 were dissolved in a mixed solution of water/acetonitrile = 1/1 and a click reaction was performed to link DBCO- ^D K8 and HApeptide-FL-SS-N3. . Subsequently purified by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA, HApeptide-FL-SS- ^D K8 was purified as a white solid. obtained as.

HApeptide-FL-SS- ^D K8, calcd for [M+H] ⁺ , 3709.8; found, 3716.1

[Experiment example 26]
(Study of intracellular introduction of HApeptide-FL-SS- ^D K8)
HApeptide-FL-SS- ^D K8 was added to the HeLa cell culture medium at a final concentration of 5 μM or 10 μM. Subsequently, 30 minutes after the addition of HApeptide-FL-SS- ^D K8, the fluorescence of FL was observed using a confocal laser microscope. In addition, dead cells were detected by PI staining and detecting PI fluorescence.

FIG. 14 is a photograph showing the results of microscopic observation. Scale bar is 80 μm. In FIG. 14, "FL" indicates a photograph in which fluorescein fluorescence was observed, "PI" indicates a photograph in which PI fluorescence was observed, and "DIC" indicates a differential interference microscope photograph. The results revealed that no FL fluorescence was detected 30 minutes after the addition of HApeptide-FL-SS- ^D K8.

This result indicates that HApeptide-FL-SS- ^D K8 did not permeate the cell membrane of the cells and diffused outside the cells.

[Experiment Example 27]
(Synthesis of DBCO-Far)
A compound represented by the following formula (22) (hereinafter sometimes referred to as "DBCO-Far") was synthesized according to the following scheme (16).

Specifically, as shown in Scheme (16), DBCO- Far was synthesized.

Fmoc deprotection was performed using 20% piperidine in N,N-dimethylformamide (DMF) for 15 minutes at room temperature. The amino acid coupling reaction was performed using Fmoc-protected amino acids (3.1 equivalents), 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU, 3.0 (eq.), 1-hydroxybenzotriazole (HOBt, 3.0 eq.) and N,N-diisopropylethylamine (DIPEA, 6.0 eq.) at room temperature. All Fmoc deprotection and coupling steps were also monitored by Kaiser test. Also, all washing procedures were performed using DMF.

First, Sieber amide resin (0.79 mmol/g) (38 mg, 30 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc deprotection and coupling reactions were repeated using Fmoc-D-Cys(Trt)-OH, Fmoc-Adox-OH, and DBCO aicd as building blocks.

Trt deprotection and cleavage from the resin was performed using trifluoroacetic acid (TFA) containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and purified with DBCO. - Got ^DC .

Subsequently, DBCO- ^DC , zinc acetate dihydrate, and trans,trans-farnesyl bromide were dissolved in a mixed solution of DMF/acetonitrile/water = 2/1/1 (0.05% TFA) to perform farnesylation. . Subsequent purification by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA afforded DBCO-Far as a transparent solid.

MALDI-TOF-MS: calcd for [M+Na] ⁺ , 865.3413; found, 865.2782

[Experiment example 28]
(Synthesis of HApeptide-FL-SS-Far)
A compound represented by the following formula (23) (hereinafter sometimes referred to as "HApeptide-FL-SS-Far") was synthesized according to the following scheme (17).

Specifically, first, Sieber amide resin (0.79 mmol/g) (76.0 mg, 60 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc-Lys(N ₂ )-OH, Fmoc-NH-ethyl-SS-propionic aicd, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc- Fmoc deprotection and coupling reactions were repeated using Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Val-OH, and acetic anhydride as building blocks. Finally, ivDde deprotection was performed and 5(6)-Carboxyfluorescein (FL) was coupled.

OtBu, tBu deprotection and cleavage from the resin was performed using TFA containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse-phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and HApeptide. -FL-SS-N3 was obtained. The amino acid sequence of HApeptide was YPYDVPDPYAK (SEQ ID NO: 5), and the molecular weight was approximately 1947.12.

Subsequently, DBCO-Far and HApeptide-FL-SS-N3 were dissolved in a mixed solution of water/acetonitrile = 1/1 and a click reaction was performed to link DBCO-Far and HApeptide-FL-SS-N3. Subsequent purification by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA yielded HApeptide-FL-SS-Far as a yellow solid. Obtained.

HApeptide-FL-SS-Far, calcd for [MH] ^- , 2991.3; found, 3001.1

[Experiment example 29]
(Study of intracellular introduction of HApeptide-FL-SS-Far)
HApeptide-FL-SS-Far was added to the HeLa cell culture medium at a final concentration of 5 μM or 10 μM. Subsequently, 30 minutes after the addition of HApeptide-FL-SS-Far, the fluorescence of FL was observed using a confocal laser microscope. In addition, dead cells were detected by PI staining and detecting PI fluorescence.

FIG. 14 is a photograph showing the results of microscopic observation. Scale bar is 80 μm. In FIG. 14, "FL" indicates a photograph in which fluorescein fluorescence was observed, "PI" indicates a photograph in which PI fluorescence was observed, and "DIC" indicates a differential interference microscope photograph. The results revealed that no FL fluorescence was detected 30 minutes after the addition of HApeptide-FL-SS-Far.

This result indicates that HApeptide-FL-SS-Far did not permeate the cell membrane of the cells and diffused outside the cells.

[Experiment example 30]
(Study of intracellular introduction of HApeptide-FL-SS-N3)
HApeptide-FL-SS-N3 was added to the HeLa cell culture medium to a final concentration of 5 μM or 10 μM. Subsequently, 30 minutes after the addition of HApeptide-FL-SS-N3, the fluorescence of FL was observed using a confocal laser microscope. In addition, dead cells were detected by PI staining and detecting PI fluorescence.

FIG. 14 is a photograph showing the results of observation using a confocal laser microscope. Scale bar is 80 μm. In FIG. 14, "FL" indicates a photograph in which fluorescein fluorescence was observed, "PI" indicates a photograph in which PI fluorescence was observed, and "DIC" indicates a differential interference microscope photograph. The results revealed that no FL fluorescence was detected 30 minutes after the addition of HApeptide-FL.

This result indicates that HApeptide-FL-SS-N3 did not permeate the cell membrane of the cells and diffused outside the cells.

[Experiment Example 31]
(Synthesis of DBCO-R8)
A compound represented by the following formula (24) (hereinafter sometimes referred to as "DBCO-R8") was synthesized according to the following scheme (18).

Specifically, as shown in Scheme (18), DBCO- R8 was synthesized.

Fmoc deprotection was performed using 20% piperidine in N,N-dimethylformamide (DMF) for 15 minutes at room temperature. The amino acid coupling reaction was performed using Fmoc-protected amino acids (3.1 equivalents), 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU, 3.0 (eq.), 1-hydroxybenzotriazole (HOBt, 3.0 eq.) and N,N-diisopropylethylamine (DIPEA, 6.0 eq.) at room temperature. All Fmoc deprotection and coupling steps were also monitored by Kaiser test. Also, all washing procedures were performed using DMF.

First, Rink amide resin (0.43 mmol/g) (46.5 mg, 20 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc deprotection and coupling reactions were repeated using Fmoc-Arg(Pbf)-OH, Fmoc-Adox-OH, and DBCO aicd as building blocks.

Pbf deprotection and cleavage from the resin was performed using trifluoroacetic acid (TFA) containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and purified with DBCO. -R8 was obtained.

[Experiment example 32]
(Synthesis of HApeptide-FL-SS-R8)
A compound represented by the following formula (25) (hereinafter sometimes referred to as "HApeptide-FL-SS-R8") was synthesized according to the following scheme (19).

Specifically, first, Sieber amide resin (0.79 mmol/g) (76.0 mg, 60 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc-Lys(N ₂ )-OH, Fmoc-NH-ethyl-SS-propionic aicd, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc- Fmoc deprotection and coupling reactions were repeated using Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Val-OH, and acetic anhydride as building blocks. Finally, ivDde deprotection was performed and 5(6)-Carboxyfluorescein (FL) was coupled.

OtBu, tBu deprotection and cleavage from the resin was performed using TFA containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse-phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and HApeptide. -FL-SS-N3 was obtained. The amino acid sequence of HApeptide was YPYDVPDPYAK (SEQ ID NO: 5), and the molecular weight was approximately 1947.12.

Subsequently, DBCO-R8 and HApeptide-FLP-SS-N3 were dissolved in a mixed solution of water/acetonitrile = 1/1 and a click reaction was performed to link DBCO-R8 and HApeptide-FL-SS-N3. Subsequent purification by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA yielded HApeptide-FL-SS-R8 as a yellow solid. Obtained.

HApeptide-FL-SS-R8, calcd for [M+H] ⁺ , 3933.9; found, 3934.2

[Experiment example 33]
(Study of intracellular introduction of HApeptide-FL-SS-R8)
HApeptide-FL-SS-R8 was added to the HeLa cell culture medium at a final concentration of 5 μM or 10 μM. Subsequently, 30 minutes after the addition of HApeptide-FL-SS-R8, FL fluorescence was observed using a confocal laser microscope. In addition, dead cells were detected by PI staining and detecting PI fluorescence.

FIG. 14 is a photograph showing the results of microscopic observation. Scale bar is 80 μm. In FIG. 14, "FL" indicates a photograph in which fluorescein fluorescence was observed, "PI" indicates a photograph in which PI fluorescence was observed, and "DIC" indicates a differential interference microscope photograph. The results revealed that 30 minutes after the addition of HApeptide-FL-SS-R8, FL fluorescence was detected only as an endosomal signal and was not detected from the cytoplasm.

This result indicates that HApeptide-FL-SS-R8 permeates the cell membrane through the endocytic pathway, remains in endosomes, and hardly enters the cytoplasm. Note that R8 has conventionally been used as a peptide that penetrates cell membranes.

[Experiment example 34]
(Synthesis of FL-SS- ^D K8Far)
A compound represented by the following formula (26) (hereinafter sometimes referred to as "FL-SS- ^D K8Far") was synthesized according to the following scheme (20).

Specifically, first, Sieber amide resin (0.79 mmol/g) (76.0 mg, 60 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc deprotection and coupling reactions were repeated using Fmoc-Lys(N ₂ )-OH, Fmoc-NH-ethyl-SS-propionic aicd, and 5(6)-Carboxyfluorescein (FL) as building blocks.

Cleavage from the resin was performed using TFA containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse-phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA, followed by FL -SS-N3 was obtained.

Subsequently, DBCO- ^D K8Far and FL-SS-N3 were dissolved in a mixed solution of water/acetonitrile = 1/1 and a click reaction was performed to link DBCO- ^D K8Far and FL-SS-N3. Subsequent purification by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA yielded FL-SS- ^D K8Far as a yellow solid. Ta.

FL-SS- ^D K8Far, calcd for [M+H] ⁺ , 2764.4791; found, 2765.6374

[Experiment example 35]
(Study of intracellular introduction of FL-SS- ^D K8Far)
FL-SS- ^D K8Far was added to the HeLa cell culture medium at a final concentration of 5 μM or 10 μM. For comparison, samples were also prepared in which fluorescein (FL) was added to the HeLa cell culture medium to a final concentration of 5 μM or 10 μM. Note that fluorescein is a compound that does not permeate cell membranes. Subsequently, FL fluorescence was observed using a confocal laser microscope.

FIG. 15 is a photograph showing the results of microscopic observation. Scale bar is 40 μm. In FIG. 15, "FL" indicates a photograph in which fluorescein fluorescence was observed, "PI" indicates a photograph in which PI fluorescence was observed, and "DIC" indicates a differential interference microscope photograph. The results revealed that FL fluorescence was detected in the cytoplasm 30 minutes after the addition of FL-SS- ^D K8Far. On the other hand, no FL fluorescence was detected from HeLa cells in which FL was added to the medium.

This result indicates that FL-SS- ^D K8Far penetrated the cell membrane of the cells, was introduced into the cytoplasm, and diffused into the cytoplasm.

FIG. 16(a) is a graph showing the results of calculating the peptide introduction rate of each compound based on the results of Experimental Example 22, Experimental Example 26, and Experimental Example 35. The peptide introduction rate was calculated based on the following formula (F1).
Peptide introduction rate (%) = number of cells in which FL fluorescence was detected from the cytoplasm/total number of cells x 100...(F1)

FIG. 16(b) is a graph showing the results of evaluating the cytotoxicity of each compound based on the results of Experimental Example 22, Experimental Example 26, and Experimental Example 35. Cell survival rate (%) was calculated based on the following formula (F2).
Cell survival rate (%) = (1-(number of cells in which PI fluorescence was detected/total number of cells)) x 100...(F2)

In FIGS. 16(a) and 16(b), the values in the graphs represent the average value ± standard deviation based on the results of more than 30 cells.

[Experiment example 36]
(Synthesis of SH2peptide-SS- ^D K8Far)
As an experimental example of intracellular introduction of a physiologically active peptide, introduction of SH2 peptide was investigated. The PI3K/Akt (Phosphoinositide 3-kinase/Protein Kinase B) pathway was selected as the target pathway. This pathway is a signal transduction pathway involved in all cellular functions such as metabolism and cell proliferation. Enhanced activity of the PI3K/Akt pathway is known to be associated with various human diseases such as cancer and neurological diseases, and is an important drug discovery target. In this experimental example, SH2 peptide, which inhibits PI3K activation by binding to the SH2 domain of PI3K, was introduced into the cytoplasm with the aim of inhibiting signal transduction of the PI3K/Akt pathway. We expected that if SH2 peptide could be efficiently introduced into the cytoplasm, phosphorylation of downstream Akt would be reduced.

First, a compound represented by the following formula (27) (hereinafter sometimes referred to as "SH2peptide-SS ^-D K8Far") was synthesized according to the following scheme (21).

Specifically, first, Rink amide resin (0.43 mmol/g) (46.5 mg, 20 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc-Lys(N ₂ )-OH, Fmoc-NH-ethyl-SS-propionic acid, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Pro-OH, Fmoc-Val-OH, Fmoc- Fmoc deprotection and coupling reactions were repeated using F ₂ Pmp-OH, Fmoc-Asp(OtBu)-OH, and acetic anhydride as building blocks.

OtBu deprotection and cleavage from the resin was performed using TFA containing 2.5% triisopropylsilane (TIPS) and 2.5% water. The crude product was subsequently precipitated with diethyl ether and purified by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA and purified with SH2peptide. -SS-N3 was obtained. The amino acid sequence of SH2peptide was DXVPMLK (SEQ ID NO: 1, X=phosphonodifluoromethylphenylalanine (F2Pmp)), and the molecular weight was about 1209.35.

Subsequently, DBCO- ^D K8Far and SH2peptide-SS-N3 were dissolved in a mixed solution of water/acetonitrile = 1/1 and a click reaction was performed to link DBCO- ^D K8Far and SH2peptide-SS-N3. Subsequent purification by reverse phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA yielded SH2peptide-SS- ^D K8Far as a white solid. Ta.

MALDI-TOF-MS: calcd for [M+2H] ²⁺ , 1641.3731; found, 1641.8861

[Experiment example 37]
(Examination of PI3K/Akt pathway inhibition by SH2peptide-SS- ^D K8Far)
FIGS. 17(a) to (c) are schematic diagrams illustrating inhibition experiments of the PI3K/Akt pathway. FIG. 17(a) shows the case where cells are not stimulated with epidermal growth factor (EGF). In this case, PI3K is not activated and downstream Akt phosphorylation is not observed. FIG. 17(b) shows the case where cells were stimulated with EGF. In this case, PI3K is activated and downstream Akt is phosphorylated. FIG. 17(c) shows the case where cells were stimulated with EGF in the presence of SH2peptide-SS- ^D K8Far. In this case, SH2peptide-SS ^-D K8Far is introduced into the cytoplasm through the cell membrane and binds to PI3K. As a result, activation of PI3K is inhibited. As a result, downstream Akt phosphorylation is also suppressed.

A PI3K/Akt pathway inhibition experiment was performed. SH2peptide-SS- ^D K8Far was added to the HeLa cell culture medium at a final concentration of 5 μM and treated for 20 minutes. Next, the HeLa cells were washed, and EGF was added to the HeLa cell culture medium at a final concentration of 50 ng/mL and treated for 5 minutes. Next, the HeLa cells were washed, and a cell lysate was used to prepare a HeLa cell lysate. Next, using SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, the HeLa cell lysate was separated by molecular weight, and Akt phosphorylation was quantitatively analyzed.

FIG. 18(a) is a photograph showing the results of quantitative analysis of Akt phosphorylation by SDS-PAGE and Western blotting. FIG. 18(b) is a graph quantifying Akt phosphorylation based on FIG. 18(a).

When HeLa cells were stimulated with EGF, phosphorylation of Akt (T308 and S473) was significantly increased (FIGS. 18(a) and (b), Lane 2). In contrast, when HeLa cells were treated with SH2peptide-SS ^-D K8Far and then stimulated with EGF, Akt phosphorylation (T308 and S473) was suppressed (Figures 18(a) and (b)). , Lane 3). In contrast, when the same experiment was performed using SH2peptide-SS-N3, no decrease in Akt phosphorylation (T308 and S473) was observed (Figures 18(a) and (b), Lane 4). .

This result indicates that SH2peptide-SS- ^D K8Far was introduced into the cytoplasm and inhibited the downstream Akt phosphorylation by inhibiting the activation of PI3K.

According to the present invention, it is possible to provide a technique for delivering a cell membrane-impermeable substance to the cytoplasm of an animal cell.

Claims (8)

A compound represented by the following formula (1).

[In formula (1), X ¹ represents a hydrogen atom, an acetyl group, or -L ² -R ² , X ² represents -NH ₂ , -OH or -L ² -R ² , and the combination of X ¹ and X ² At least one is -L ² -R ² , and L ¹ and L ² each independently represent one or more -C-, -O-, -CO-, -NH-, -S- or -S Represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be substituted with -S-, R ¹ is a prenyl group having 1 to 5 isoprene units, or a saturated or unsaturated group having 10 to 30 carbon atoms. It represents an acyl group or a saturated or unsaturated alkyl group having 10 to 30 carbon atoms, and R ² represents a binding functional group or a monovalent group derived from a cell membrane-impermeable substance with a molecular weight of 100 to 5000. where m and n each independently represent an integer of 0 to 20, and p and q each independently represent an integer of 1 to 4. ] The compound according to claim 1, wherein L ² in the formula (1) contains a -S-S- group. The compound according to claim 1 or 2, wherein the sum of m and n in the formula (1) is 5 or more. X ¹ in the formula (1) is -L ² -R ² and n is 7 or less, or X ² in the formula (1) is -L ² -R ² and m is 7 or less The compound according to any one of claims 1 to 3, which is The compound according to any one of claims 1 to 4, which when brought into contact with a cell, penetrates the cell membrane of the cell and is introduced into the cell. The compound according to any one of claims 1 to 5, wherein the cell membrane-impermeable substance having a molecular weight of 100 to 5,000 is a peptide. A method for modifying a cell membrane-impermeable substance with a molecular weight of 100 to 5000 into a cell-permeable substance, the method comprising:
A method comprising the step of bonding a group represented by the following formula (2) or a group represented by the following formula (2') to the cell membrane-impermeable substance.

[In formula (2) and formula (2'), L ¹ and L ² each independently represent one or more -C-, -O-, -CO-, -NH-, -S- or - Represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be substituted with SS-, R ¹ is a prenyl group having 1 to 5 isoprene units, or a saturated or unsaturated group having 10 to 30 carbon atoms. represents an acyl group or a saturated or unsaturated alkyl group having 10 to 30 carbon atoms, m and n each independently represent an integer of 0 to 20, and p and q each independently represent an integer of 1 to 4. represent. Further, in formula (2), X ³ represents -NH ₂ or -OH, and in formula (2'), X ⁴ represents a hydrogen atom or an acetyl group. ] A method of introducing a cell membrane-impermeable substance with a molecular weight of 100 to 5000 into the cytoplasm of a target cell,
A method comprising the step of contacting the target cell with the substance to which a group represented by the following formula (2) or a group represented by the following formula (2') is bound.

[In formula (2) and formula (2'), L ¹ and L ² each independently represent one or more -C-, -O-, -CO-, -NH-, -S- or - Represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be substituted with SS-, R ¹ is a prenyl group having 1 to 5 isoprene units, or a saturated or unsaturated group having 10 to 30 carbon atoms. represents an acyl group or a saturated or unsaturated alkyl group having 10 to 30 carbon atoms, m and n each independently represent an integer of 0 to 20, and p and q each independently represent an integer of 1 to 4. represent. In formula (2), X ³ represents -NH ₂ or -OH, and in formula (2'), X ⁴ represents a hydrogen atom or an acetyl group. ]

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