JP2021013373A - Microbial membrane penetrating agents, conjugates, methods for delivering compounds to microbial cells using the same, bactericidal agents, antibacterial agents and labeling agents - Google Patents

Abstract

To provide a microbial membrane penetrating agent that can penetrate a membrane of microbe such as colibacillus with high efficiency and low toxicity, and introduce compounds of interest into microbial cells.SOLUTION: Disclosed is a microbial membrane penetrating agent comprising at least one of a first membrane penetrating peptide and a second membrane penetrating peptide, where the first membrane penetrating peptide comprises 4-26 of an independently selected amino acid. The microbial membrane penetrating agent can efficiently penetrate the membrane of bacteria such as Escherichia coli exhibiting low cytotoxicity. Bactericidal agents utilizing the penetrating agent of the invention can be used for antiseptic, bactericidal, sterilizing, agrohorticultural, medical or disinfecting purpose.SELECTED DRAWING: Figure 1

Description

The present disclosure relates to microbial membrane penetrants, complexes, methods of delivering compounds to microbial cells using the same, fungicides, antibacterial agents and labeling agents.

Membrane-permeable substances are effective molecular tools that enable the introduction of functional molecules and drugs into cells, and are used in various research fields related to biochemistry and pharmacy, and their importance and necessity are increasing. There is. The search for substances that permeate membranes with high efficiency in animal cells is being actively carried out, and for example, there are many reports of peptides (cell-penetrating peptides, CPP) that permeate animal cell membranes. As an example of CPP, a method of incorporating a molecule into a cell by binding an arginine oligomer and a lysine oligomer as a membrane-permeable peptide to the molecule has been reported (Patent Document 1).

On the other hand, there are few studies on membrane-permeable substances for prokaryotic cells such as microorganisms. Animal cells and microbial cells have very different membrane structures. Specifically, animal cells have only a cell membrane, whereas microbial cells have a peptidoglycan layer on the outside of the cell membrane. In addition, Gram-negative bacteria have an outer membrane on the outside of the peptidoglycan layer. Considering these differences in structure and the multi-membrane structure of microorganisms, that is, the structure that blocks the permeation of substances, it is unlikely that there is a simple commonality between animal cell membrane-permeable substances and microbial membrane-permeable substances. , Microbial membrane permeable materials need to be developed independently of animal cell membrane permeable materials.

For example, known as cell-permeable peptides of animal cells (RFF) ₃ (9 amino acid peptides containing 3 repetitions of arginine, phenylalanine and phenylalanine), K9 (peptide consisting of 9 lysines) and R9 (9 arginines). (Peptide consisting of) has a low efficiency of introducing a functional substance into microbial cells, and the efficiency of introduction into Escherichia coli (measured value) is only 4.0%, 40.3% and 68.2%, respectively. Moreover, until now has been considered a good introduction rate against prokaryotic cells (KFF) 3 K _(Lysine, 9 amino acid peptide containing three repetitions of phenylalanine and phenylalanine) is also introduced efficiency (measured value) to E. coli It was about 12.8%.

There is also a report on a cell-permeable peptide (CPP) for Escherichia coli (Non-Patent Document 1), but this is cell permeability after pretreating the E. coli cells to be introduced into a state in which substances can be easily taken up (competent cells). A substance bound to a peptide is introduced.

Special Table No. 55-500053

K. Oikawa, 4 others, ACS Omega, American Chemical Society (USA), December 2018, Volume 3, Issue 12, p.16489-16499

Therefore, the present disclosure describes a microbial membrane permeating agent, a complex, and a microorganism using the same, which can permeate the cell membrane of a microorganism such as Escherichia coli with high efficiency and low toxicity and introduce a target compound into the cell of the microorganism. It is an object of the present invention to provide a method for delivering a compound to a cell, a bactericidal agent, an antibacterial agent, and a labeling agent.

As a result of diligent studies to solve the above problems, the present inventor has found that such a microbial membrane permeating agent efficiently permeates the cell membrane of bacteria such as Escherichia coli and has low cytotoxicity, and completes the present disclosure. I came to do it.

The present disclosure includes, for example, the following aspects:

[1] A first membrane-permeable peptide containing 4 to 26 independently selected amino acid residues of formula (I) in succession, and a second containing amino acid residues of formula (II). A microbial membrane-permeable agent containing at least one of the membrane-permeable peptides of.

[In formulas (I) and (II), n is an independently integer of 1 to 3, and the amino acid residue may be either (R) -form or (S) -form. In formula (II), m is an integer of 1 to 5, and R ¹ and R ² independently represent hydrophobic substituents. In formulas (I) and (II), the site represented by * represents a binding site with an adjacent site. ]
[2] The first membrane-permeable peptide is (S) -2,5-diaminopentanoic acid (Orn), (R) -2,5-diaminopentanoic acid (Orn), (S) -2,4- Diaminobutanoic acid (Dab), (R) -2,4-diaminobutanoic acid (Dab), (S) -2,3-diaminopropionic acid (Dap) and (R) -2,3-diaminopropionic acid (Dap) The microbial membrane penetrant according to [1], which comprises a membrane-permeable peptide consisting of 4 to 26 consecutive amino acid residues independently selected from the group consisting of).
[3] The microbial membrane-permeable agent according to [1] or [2], wherein the first membrane-permeable peptide contains at least four consecutive Orn or Dab.
[4] The microbial membrane penetrant according to any one of [1] to [3], wherein the first membrane-permeable peptide comprises 5 to 26 consecutive Orn or Dab.
[5] The first membrane-permeable peptide is 5,6,7,8,9,10,11 or 12 consecutive (S) -Orn or 5,6,7,8,9,10, The microbial membrane permeating agent according to any one of [1] to [4], which comprises 11 or 12 consecutive (S) -Dabs.
[6] The microbial membrane permeating agent according to any one of [1] to [5], wherein the first membrane-permeable peptide comprises 6 to 26 consecutive same amino acid residues.
[7] The first membrane-permeable peptide or the second membrane-permeable peptide contains one or several lysine (Lys) residues or arginine (Arg) in addition to the amino acid residues of the formula (I). ) The microbial membrane permeating agent according to any one of [1] to [6], which comprises a residue.
[8] The first membrane-permeable peptide has a plurality of blocks consisting of the 4 to 26 amino acid residues of the formula (I) independently selected, and at least between the plurality of blocks. The microbial membrane permeating agent according to any one of [1] to [7], wherein one or two or more amino acid residues other than the amino acid residue of the formula (I) are continuously contained in one place.
[9] Amino acid residues other than the amino acid residue of the formula (I) include a lysine residue having a labeled molecule as a substituent or an arginine (Arg) residue having a labeled molecule as a substituent. , [8].
[10] The microorganism is at least one selected from the group consisting of prokaryotes including eubacteria and archaea, and eukaryotes including microalgae, protists, fungi and slime molds, [1] to The microbial membrane permeating agent according to any one of [9].
[11] The microbial membrane permeating agent according to any one of [1] to [10], wherein the microorganism is a bacterium belonging to the genus Enterobacter, Synechocystis, Pseudomonas, Acinetobacter or Bacillus.
[12] A complex containing the microbial membrane permeating agent according to any one of [1] to [11] and a compound.
[13] The compound is selected from the group consisting of proteins, peptides, nucleic acids, peptide nucleic acids, inorganic substances, organic metal compounds, organic compounds, macromolecules, nanoparticles, molecular aggregates including micelles and liposomes, sugars and labeling substances. The complex according to [12], which comprises at least one of these.
[14] The complex according to [12] or [13], wherein the microbial membrane permeating agent and the compound are linked directly or via a linker.
[15] The complex according to [14], wherein the linker consists of 1 to 10 consecutive amino acid residues.
[16] A method for delivering a compound to a microbial cell, which comprises contacting the complex containing the microbial membrane permeating agent according to any one of [1] to [11] with the microbial cell.
[17] A group in which the compound comprises a protein, a peptide, a nucleic acid, a peptide nucleic acid, an inorganic substance, an organic metal compound, an organic compound, a polymer, nanoparticles, a sugar and a labeling substance, and a molecular aggregate containing micelles and liposomes. The method according to [16], which comprises at least one selected from.
[18] The microorganism is at least one selected from the group consisting of prokaryotes including eubacteria and archaea, and eukaryotes including microalgae, protists, fungi and slime molds, [16] or The method according to [17].
[19] The method according to any one of [16] to [18], wherein the microorganism is a bacterium of the genus Enterobacter, Synechocystis, Pseudomonas, Acinetobacter or Bacillus.
[20] A bactericidal agent containing the microbial membrane permeating agent according to any one of [1] to [11].
[21] An antibacterial agent containing the microbial membrane permeating agent according to any one of [1] to [11].
[22] A labeling agent containing the microbial membrane permeating agent according to any one of [1] to [11].

According to the present disclosure, a microbial membrane permeating agent or complex capable of permeating the cell membrane of a microorganism such as Escherichia coli with high efficiency and low toxicity and introducing the target compound into the cell of the microorganism was used. Methods of delivering compounds to microbial cells, bactericidal agents, antibacterial agents and labeling agents are provided.

It is a figure which shows the structure of the complex Dab9-K-FAM and Orn9-K-FAM of this disclosure. It is a fluorescence microscope image (top) and a cell photograph (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in Escherichia coli, which is a Gram-negative bacterium. It is the result of flow cytometry showing the logarithm of viable cells into which the complex (Dab9-K-FAM) of the present disclosure has been introduced. It is the result of flow cytometry showing the logarithm of viable cells into which the complex (Dab9-K-FAM) of the present disclosure has been introduced. Fluorescence micrographs (top) and cell photographs (bottom) showing membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in the Gram-negative bacterium Yersinia bercovieri. A fluorescence microscopic image (top) and a cell photograph (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in the Gram-positive bacterium Bacillus megaterium are shown. It is a graph which shows the membrane permeability (black-painted symbol) and the ratio of dead cells (white symbol) of various membrane-permeable peptides. Fluorescence micrographs (top) and cell photographs (bottom) showing membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in the Gram-negative bacterium Yersinia bercovieri. It is a fluorescence microscopic image (top) and a cell photograph (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in the gram-negative bacterium Serratia grimesii. It is a fluorescence microscope image (top) and a cell photograph (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in the Gram-negative bacterium Trabulsiella guamensis. FIG. 3 is a fluorescence micrograph (top) and a cell photograph (bottom) showing the membrane permeation of the complex (Dab9-K-FAM) of the present disclosure in Enterobacter hormaechei, which is a Gram-negative bacterium. Fluorescence microscopic images (top) and cell photographs (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in the Gram-negative bacterium Proteus hauseri. It is a fluorescence microscope image (top) and a cell photograph (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in Acinetobacter radioresistens, which is a Gram-negative bacterium. It is a fluorescence microscopic image (top) and a cell photograph (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in Pseudomonas fluorescens, which is a Gram-negative bacterium. It is a fluorescence microscope image (top) and a cell photograph (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in Synechocystis PCC6803, which is a Gram-negative bacterium. Fluorescence microscopic images (top) and cell photographs (bottom) showing the membrane permeability of the complexes (Dab9-K-FAM and Dab4-K (-FAM) -Dab5) of the present disclosure in Escherichia coli, which is a Gram-negative bacterium. .. It is a figure which shows the structure of the complex (Dab4-K (-FAM) -Dab5) of this disclosure. Fluorescence microscope images (top) and cell photographs (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in Acinetobacter radioresistens, which is a Gram-negative bacterium. Fluorescence micrographs (top) and cell photographs (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in Enterobacter hormaechei, a Gram-negative bacterium. Fluorescence microscope images (top) and cell photographs (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in Escherichia coil, which is a Gram-negative bacterium. Fluorescence micrographs (top) and cell photographs (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in the Gram-negative bacterium Pantoea agglomerans. Fluorescence microscopic images (top) and cell photographs (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in the Gram-negative bacterium Proteus hauseri. FIG. ₃ is a fluorescence microscopic image (top) and a cell photograph (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in Pseudomonas fluorescens, which is a Gram-negative bacterium. Fluorescence microscopic images (top) and cell photographs (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in the Gram-negative bacterium Serratia grimesii. FIG. ₃ is a fluorescence micrograph (top) and a cell photograph (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in the Gram-negative bacterium Trabulsiella guamensis. FIG. 3 is a photomicrograph showing the temporal and concentration-dependent cytotoxicity of the complex Dab9-K-FAM of the present disclosure to normal human umbilical vein endothelial cells. FIG. 3 is a photomicrograph showing the temporal and concentration-dependent cytotoxicity of the complex Orn9-K-FAM of the present disclosure to normal human umbilical vein endothelial cells. It is a figure which shows the structure of the complex ((DabFF) 3-K-FAM) of this disclosure.

Hereinafter, the present disclosure will be described in detail.

In the present disclosure, the membrane-permeable peptide means a peptide having the ability to permeate the cell membrane of a microorganism and enter the cells.
In the present disclosure, "Dap" may be referred to as "Dpr" and "Dab" may be referred to as "Dbu".
In the present disclosure, "Dab9-K-FAM" and "(Dab) 9K-FAM" are the same molecule.
In the present disclosure, "Orn9-K-FAM" and "(Orn) 9K-FAM" are the same molecule.

The concept of a disinfectant in the present disclosure includes not only what is generally called a disinfectant, but also the concept of a so-called sterilizer, disinfectant, and disinfectant.
In the present disclosure, the concept of an antibacterial agent includes not only what is generally called an antibacterial agent, but also the concept of a so-called antibacterial agent, bacteriostatic agent, antifungal agent and the like.

In the present disclosure, the "microbial membrane permeating agent" means a "permeating agent that permeates the cell membrane of a microorganism and the peptidoglycan layer existing on the cell membrane of the microorganism". The cell membrane of the microorganism also includes a spore shell and a spore cell membrane.

≪Microbial membrane penetrant≫
The present disclosure relates to novel microbial membrane permeable agents consisting of membrane permeable peptides and their uses.
The microbial permeabilizers of the present disclosure are a first membrane-permeable peptide containing 4 to 26 independently selected amino acid residues of formula (I) in succession, and an amino acid residue of formula (II). Includes at least one of the group-containing second membrane-permeable peptides.

In formulas (I) and (II), n is independently an integer of 1 to 3, and the amino acid residue may be either the (R) form or the (S) form. In formula (II), m is an integer of 1 to 5, and R ¹ and R ² independently represent hydrophobic substituents. In formulas (I) and (II), the site represented by * represents a binding site with an adjacent site.

By having the above-mentioned constitution, the microbial membrane permeating agent of the present disclosure can permeate the cell membrane of a microorganism such as Escherichia coli with high efficiency and low toxicity, and can introduce the target compound into the cell of the microorganism.

The microbial membrane permeable agent of the present disclosure may contain other components other than the membrane permeable peptide as long as the effects of the present disclosure are not impaired. Examples of other components include stabilizers, preservatives, colorants, preservatives, thickeners, nutritional supplements, fungicides, buffers, emulsifiers, organic solvents and the like.

[Membrane-permeable peptide]
The membrane-permeable peptides of the present disclosure include a first membrane-permeable peptide containing 4 to 26 independently selected amino acid residues of formula (I) in succession, and an amino acid of formula (II). At least one of the second membrane-permeable peptides containing residues.

(First membrane-permeable peptide)
The first membrane-permeable peptide contains 4 to 26 independently selected amino acid residues of formula (I) in succession.
In formula (I), n is an independently integer of 1 to 3, and the amino acid residue may be either (R) -form or (S) -form.
In formula (I), the site represented by * represents a binding site with an adjacent site. The adjacent site refers to a structure adjacent to an amino acid residue of the formula (I) such as an adjacent amino acid residue, a terminal protecting group, an N-terminal hydrogen atom, a C-terminal hydroxy group, or a linker described later. Represent.

Hereinafter, "a microbial membrane-permeable peptide containing 4 to 26 independently selected amino acid residues of formula (I)" and "a membrane-permeable peptide containing amino acid residues of formula (II)" are described below. Are collectively referred to as the "microbial membrane-permeable peptide of the present disclosure".
Hereinafter, "a microbial membrane-permeable peptide containing 4 to 26 independently selected amino acid residues of the formula (I)" is also referred to as a "first microbial membrane-permeable peptide".
Hereinafter, the "membrane-permeable peptide containing the amino acid residue of the formula (II)" is also referred to as a "second microbial membrane-permeable peptide".

For example, when n = 1, the amino acid residue of the formula (I) is the (R) or (S) form of 2,3-diaminopropionic acid (Dap).
For example, when n = 2, the amino acid residue of the formula (I) is the (R) or (S) form of 2,4-diaminobutanoic acid (Dab).
For example, when n = 3, the amino acid residue of the formula (I) is the (R) or (S) form of 2,5-diaminopentanoic acid (Orn).

The first membrane-permeable peptide has (S) -2,5-diaminopentanoic acid (Orn), (R) -2 from the viewpoint of permeating the cell membrane of microorganisms such as Escherichia coli with higher efficiency and lower toxicity. , 5-Diaminopentanoic acid (Orn), (S) -2,4-diaminobutanoic acid (Dab), (R) -2,4-diaminobutanoic acid (Dab), (S) -2,3-diaminopropion It preferably consists of 4 to 26 contiguous amino acid residues independently selected from the group consisting of acid (Dap) and (R) -2,3-diaminopropionic acid (Dap).

In the present disclosure, when it is described that a predetermined number of amino acid residues selected from a plurality of options are contiguous, each amino acid residue contained in the contiguous amino acid residues is selected from the plurality of options. As long as they are the same, they may be the same or different from each other, and as an example, the case where the same amino acid residues are consecutive in a predetermined number is included.

The amino acid sequence of the first membrane-permeable peptide may be, for example, SEQ ID NO: 1 below.
X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25- X26 (SEQ ID NO: 1)

In SEQ ID NO: 1,
X1 to X3 independently contain (S) -2,5-diaminopentane acid (Orn), (R) -2,5-diaminopentane acid (Orn), and (S) -2,4-diaminobutane. With acid (Dab), (R) -2,4-diaminobutanoic acid (Dab), (S) -2,3-diaminopropionic acid (Dap) or (R) -2,3-diaminopropionic acid (Dap) Yes,
X4 to X26 do not exist independently of each other, or (S) -2,5-diaminopentanoic acid (Orn), (R) -2,5-diaminopentanoic acid (Orn), (S)- 2,4-Diaminobutanoic acid (Dab), (R) -2,4-diaminobutanoic acid (Dab), (S) -2,3-diaminopropionic acid (Dap) or (R) -2,3-diamino Propionic acid (Dap).

The first membrane-permeable peptide is not particularly limited as long as it is a peptide containing 4 to 26 independently selected amino acid residues of the formula (I) in succession.
It is preferably a peptide containing 5 to 26 independently selected amino acid residues of the formula (I) in succession.
More preferably, it is a peptide containing 6 to 26 independently selected amino acid residues of the formula (I) in succession.
More preferably, it is a peptide containing 6 to 20 independently selected amino acid residues of formula (I) in succession.

The stereoisomers of the amino acid residues constituting the first membrane-permeable peptide are not particularly limited, and all the amino acid residues constituting the membrane-permeable peptide are in the (R) form or the (S) form. It may be present, or the (R) body and the (S) body may be mixed.

From the viewpoint of further improving the membrane permeability, the first membrane-permeable peptide is preferably composed of 4 to 26 consecutive amino acid residues, and 5 to 26 consecutive same amino acid residues. It is more preferably composed of, more preferably 6 to 26 consecutive same amino acid residues, and particularly preferably 20 consecutive same amino acid residues.

The first membrane-permeable peptide may be a peptide having an arbitrary amino acid sequence as long as it contains the amino acid residue of the above formula (I) and retains the microbial membrane permeability.

The first membrane-permeable peptide is used from the viewpoint of further improving membrane permeability.
It preferably contains at least 4 consecutive Orn, Dab or Dap.
More preferably, it contains at least 4 consecutive Orn or Dab.
More preferably, it contains 5 to 26 consecutive Orn or Dab.
It is particularly preferred to include 6 to 26 consecutive Orn or Dab.

The first membrane-permeable peptide is used from the viewpoint of further improving membrane permeability.
It consists of 5, 6, 7, 8, 9, 10, 11 or 12 consecutive (S) -Orn,
It consists of 5, 6, 7, 8, 9, 10, 11 or 12 consecutive (S) -Dabs,
Alternatively, it is preferably composed of 9 consecutive (S) -Dap.

The first membrane-permeable peptide consists of 6, 7, 8, 9, 10, 11 or 12 consecutive (S) -Orns, or 6, 7, 8, 9, 10, 11 or 12 It is more preferably composed of a number of consecutive (S) -Dabs.

The first membrane-permeable peptide consists of 8, 9, 10, 11 or 12 contiguous (S) -Orn, or 6, 7, 8, 9, 10, 11 or 12 contiguous. It is more preferably composed of (S) -Dab.

As an example, the amino acid sequences of the Dab9 dimer (also referred to as Dab9 or (Dab) 9) and the Orn9 dimer (also referred to as Orn9 or (Orn) 9) are shown below.
Dab9: Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab (SEQ ID NO: 2)
Orn9: Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn (SEQ ID NO: 3)

In addition, the amino acid sequences of the other first membrane-permeable peptides; Orn 6-12 mer, Dab 6-12, 15, 19 mer and Dap 9 mer are shown below.
Orn6: Orn-Orn-Orn-Orn-Orn-Orn (SEQ ID NO: 7)
Orn7: Orn-Orn-Orn-Orn-Orn-Orn-Orn (SEQ ID NO: 8)
Orn8: Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn (SEQ ID NO: 9)
Orn10: Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn (SEQ ID NO: 10)
Orn11: Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn (SEQ ID NO: 11)
Orn12: Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn (SEQ ID NO: 12)
Dab4: Dab-Dab-Dab-Dab (SEQ ID NO: 20)
Dab5: Dab-Dab-Dab-Dab-Dab (SEQ ID NO: 21)
Dab6: Dab-Dab-Dab-Dab-Dab-Dab (SEQ ID NO: 13)
Dab7: Dab-Dab-Dab-Dab-Dab-Dab-Dab (SEQ ID NO: 14)
Dab8: Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab (SEQ ID NO: 15)
Dab10: Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab (SEQ ID NO: 16)
Dab11: Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab (SEQ ID NO: 17)
Dab12: Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab (SEQ ID NO: 18)

Dap9: Dap-Dap-Dap-Dap-Dap-Dap-Dap-Dap-Dap (SEQ ID NO: 19)

Dab15: Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab (SEQ ID NO: 24)
Dab19: Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab-Dab (SEQ ID NO: 25)

The three isomers in the above-mentioned specific example of the first membrane-permeable peptide may be all of Orn, Dab and Dap in the (R) form or the (S) form, or the (R) form and the (S) form. ) A body may be mixed, but it is preferable that all of Orn, Dab and Dap are (S) bodies.

In one embodiment, the first membrane-permeable peptide has a plurality of blocks consisting of the 4 to 26 amino acid residues of the formula (I) independently selected, and at least between the plurality of blocks. It may be an embodiment in which one or two or more amino acid residues other than the amino acid residue of the above formula (I) are continuously contained in one place. In this case, the amino acid residue other than the amino acid residue of the formula (I) preferably contains a lysine (Lys) residue or an arginine (Arg) residue, and a lysine residue having a labeled molecule as a substituent. More preferably, it contains a group or an arginine (Arg) residue. Examples of the labeling molecule include the same labeling molecules as those exemplified in the complex of the present disclosure described later.

In one embodiment, the first membrane-permeable peptide is, for example, lysine (Lys) in which the tag molecule 5 (6) -carboxyfluorescein (FAM) is substituted as a substituent at the terminal of the Dab9 mer, shown below. It may be an amino acid sequence having one residue.
Dap9 (FAM): Dap-Dap-Dap-Dap-Dap-Dap-Dap-Dap-Dap-K (-FAM) (SEQ ID NO: 22)

In one aspect, the first membrane-permeable peptide is, for example, substituted with the labeling molecule 5 (6) -carboxyfluorescein (FAM) as a substituent between the Dab tetramer and the Dab pentamer shown below. It may be an amino acid sequence mediated by one residue of lysine (Lys).
Dab4-K (-FAM) -Dab5: Dab-Dab-Dab-Dab-K (-FAM) -Dab-Dab-Dab-Dab-Dab (SEQ ID NO: 23)

(Second membrane-permeable peptide)
The second membrane-permeable peptide comprises an amino acid residue of formula (II).
In formula (II), n is an independently integer of 1 to 3, and the amino acid residue may be either (R) -form or (S) -form.
In formula (II), m is an integer of 1 to 5, and R ¹ and R ² independently represent hydrophobic substituents.
In formula (II), the site represented by * represents a binding site with an adjacent site. The adjacent site refers to a structure adjacent to an adjacent amino acid residue, a terminal protecting group, an N-terminal hydrogen atom, a C-terminal hydroxy group, or an amino acid residue of formula (II) such as a linker described later. Represent.

In the formula (II), n is independently an integer of 1 to 3, and from the viewpoint of further improving the membrane permeability, it is preferably an integer of 2 to 3, and more preferably 2.
In the formula (II), m is an integer of 1 to 5, and from the viewpoint of further improving the membrane permeability, it is preferably an integer of 2 to 4, and more preferably an integer of 2 to 3.

In formula (II), R ¹ and R ² each independently represent a hydrophobic substituent.

The hydrophobic substituent represented by R ^1, an amino acid residue comprising NH and C = O ^{R 1} is located at both ends of the carbon atoms substituted ^{(NH-C (R 1)} -C (= O)) Means that is an amino acid residue derived from a hydrophobic amino acid.
The hydrophobic substituent represented by R ^2, an amino acid residue comprising NH and C = O ^{R 2} are located at both ends of the carbon atoms substituted ^{(NH-C (R 2)} -C (= O)) Means that is an amino acid residue derived from a hydrophobic amino acid.

The hydrophobic amino acid means an amino acid having a hydrophobic index (hi) of -2 or more. Specifically, the hydrophobic amino acids include glycine (hi = -0.4), tryptophan (hi = 0.9), methionine (hi = 1.9), proline (hi = -1.6), and phenylalanine (hi). = 2.8), alanine (hi = 1.8), valine (hi = 4.2), leucine (hi = 3.8), isoleucine (hi = 4.5) and the like. Among the above, the hydrophobic amino acid preferably contains phenylalanine.

The stereoisomers of the amino acid residues constituting the second membrane-permeable peptide are not particularly limited, and all the amino acid residues constituting the membrane-permeable peptide are in the (R) form or the (S) form. It may be present, or the (R) body and the (S) body may be mixed.

The stereoisomers in the above-mentioned second membrane-permeable peptide may be all of Orn, Dab and Dap in the (R) form or the (S) form, or the (R) form and the (S) form. May be mixed, but it is preferable that all of Orn, Dab and Dap are in the (S) form.

The second membrane-permeable peptide may be a peptide having an arbitrary amino acid sequence as long as it contains the amino acid residue of the above formula (II) and retains the microbial membrane permeability.

The second membrane-permeable peptide is used from the viewpoint of further improving membrane permeability.
It preferably contains at least one Orn, Dab or Dap.
More preferably, it contains at least two Orn or Dab.
It is more preferable to include 3 Dab.

In one embodiment, the second membrane-permeable peptide is, for example, a sequence in which the repeating unit consisting of a Dab1 mer and a phenylalanine dimer shown below is consecutive three times, and a labeled molecule as a substituent 5 (6) It may be an amino acid sequence having one residue of lysine (Lys) substituted with -carboxyfluorescein (FAM).
(DabFF) 3-K-FAM: Dab-FF-Dab-FF-Dab-FFK (-FAM) (SEQ ID NO: 26)

The microbial membrane-permeable agent of the present disclosure may contain at least one of a first membrane-permeable peptide and a second membrane-permeable peptide, and the first membrane-permeable peptide and the second membrane-permeable peptide may be contained. May include both. When the microbial membrane-permeable agent contains both a first membrane-permeable peptide and a second membrane-permeable peptide, both may be mediated by other amino acid residues described below.

The membrane-permeable peptide of the present disclosure may contain other amino acid residues other than the amino acid residue of formula (I) and the amino acid residue of formula (II) as long as it does not affect the microbial membrane permeability. ..
When the other amino acid residue is contained, the other amino acid residue is not particularly limited, but is preferably 1 to 5 arbitrary amino acid residues.
The other amino acid residue is preferably, for example, a lysine (Lys) residue or an arginine (Arg) residue.
The positions of the other amino acid residues are the terminal of the membrane-permeable peptide, between the amino acid residues of the plurality of formulas (I), between the amino acid residues of the plurality of formulas (II), and the formula (I). It may be between the amino acid residue of the formula (II) and the amino acid residue of the formula (II). Other amino acid residues may have a labeled molecule or the like as a substituent.

The membrane-permeable peptides of the present disclosure can be prepared by methods known in the art. For example, solid phase peptide synthesis methods and gene recombination methods for introducing unnatural amino acids into polypeptides (eg, Taira, H. et al., Biochem. Biophys. Res. Commun. 2008, 374, 304-308. , Noren, CJ et al., Science 244: 182-188, 1989, Patent No. 4917044, etc.) are available. Dab, Orn and Dap are commercially available.

The membrane-permeable peptide of the present disclosure can be easily synthesized in a high yield by a conventional solid-phase synthesis method, and can be mass-produced.

The microbial membrane permeating agent according to the present disclosure can introduce the compound into the cells of the microorganism by connecting with the compound to be introduced and bringing it into contact with the microorganism. That is, the microbial membrane penetrant functions as a vector or carrier that carries various substances into the microbial cells. The compound to be introduced is a compound other than the microbial membrane permeating agent. The compound to be introduced is not particularly limited as long as it is a compound that can be delivered by the microbial membrane permeating agent.

The target "microorganism" refers to various types of microorganisms including prokaryotes (eg, eubacteria, archaea, etc.) and eukaryotes (eg, microalgae, protists, fungi, slime, etc.). ..
Bacteria (eubacteria) are classified into gram-negative bacteria and gram-positive bacteria depending on whether or not they have an outer membrane outside the cell membrane. The microbial membrane penetrants according to the present disclosure are gram-negative bacteria and gram-positive bacteria. Applicable to both bacteria.

Examples of the bacterium include bacteria of the genus Enterobacter, Synechocystis, Pseudomonas, Acinetobacter or Bacillus. Examples of the bacteria of the family Enterobacter include bacteria belonging to the genus Yersinia, Serratia, Trouble Sierra, Enterobacter, Proteus and the like. Examples of the bacterium belonging to the genus Synechocystis include Synechocystis PCC6803. Examples of the bacterium belonging to the genus Pseudomonas include Pseudomonas fluorescence. Examples of the bacterium belonging to the genus Acinetobacter include Acinetobacter radioresistence and the like. Bacteria belonging to the genus Bacillus include Bacillus megaterium and the like.

The microbial membrane permeating agent according to the present disclosure can permeate the cell membrane of a microorganism such as Escherichia coli with high efficiency and low toxicity. Therefore, the microbial membrane permeating agents of the present disclosure are used for basic research on the dynamics of microorganisms, for searching for antibacterial substances, for drug efficacy evaluation, for antibacterial, for sterilization (for sterilization), for safety evaluation, for medical use, and for environmental measurement. It can be deployed for various purposes.

≪Complex≫
In another aspect, the present disclosure provides a complex comprising the microbial membrane permeating agent of the present disclosure and a compound. The compound is not particularly limited as long as it is a compound that permeates the cell membrane of a microorganism and is introduced into the cells.
Compounds to be introduced into cells include, for example, proteins, peptides, nucleic acids, peptide nucleic acids, inorganic substances, organic metal compounds, organic compounds, macromolecules, nanoparticles, sugars and labeling substances, and molecules including one of micelles and liposomes. It is preferable to include at least one selected from the group consisting of aggregates. These compounds can be prepared by methods known in the art or available as commercial products.

Examples of the protein include enzymes, receptors, antibodies, fluorescent proteins, cytoskeleton-forming proteins, amyloid-forming proteins and the like.
Examples of the peptide include redox active peptides such as antigens, hormones, antibacterial peptides, signal peptides, inhibitor peptides, and glutathione; organella target peptides; and the like.
Examples of the nucleic acid include DNA, RNA, hybrid nucleic acid, aptamer and the like.
Examples of peptide nucleic acids include peptide DNA and peptide RNA.
Examples of the inorganic compound, the organic compound, the organometallic compound and the nanoparticles include known compounds that can be introduced into the cell.
Examples of the polymer include polyethylene glycol, dendrimer and the like.
Examples of sugars include polysaccharides, disaccharides, monosaccharides, cyclodextrin and the like.
Examples of the labeling substance include 5 (6) -carboxyfluorescein (FAM) and the like.
Examples of the molecular aggregate include micelles and liposomes. In the present disclosure, the "molecular assembly" means an assembly in which molecules such as lipids are aggregated.

The method for linking the microbial membrane permeating agent and the compound is not particularly limited, but for example, (1) direct linking (that is, covalent bond); (2) linking via a linker; (3) hydrogen bond, hydrophobicity. Linkage by intermolecular interactions such as sexual interaction, electrostatic interaction, coordination bond; (4) Anti-antibody binding, protein ligand interaction (eg, avidin and biotin) in which the intermolecular interaction acts in a complex manner. Interaction, etc.), linkage by multiple helix formation of DNA, etc.;

In one aspect, the method of linking the microbial membrane permeating agent and the compound is to use an amino group of an amino acid residue of a membrane penetrating peptide or an amino acid residue of a linker and a reactive functional group (active ester group such as NHS ester, epoxy). A method of linking the two may be carried out by allowing a compound having a group (group, aldehyde group, etc.) to act on the compound.

In one aspect, the linking position between the microbial membrane permeable agent and the compound may be on the C-terminal side of the membrane permeable peptide in the microbial membrane permeable agent.

As one aspect, a method of linking a microbial membrane permeating agent and a compound is a method of linking a microbial membrane permeating agent containing a membrane permeable peptide in which a carboxylic acid group is converted into an active ester by an activator or the like, and a compound having a reactive functional group. And, may be a method of connecting the two by acting.

When the microbial membrane permeating agent and the compound are linked via a linker, a known linker sequence can be applied to the linker sequence. For example, as the linker sequence, 1 to 20 consecutive amino acid residues, preferably 1 to 10 consecutive amino acid residues, and more preferably 1 to 6 consecutive amino acid residues can be applied. Known amino acids can be applied as the type of amino acid forming the linker, and examples thereof include lysine residue and arginine residue from the viewpoint of further maintaining the membrane permeability of the microbial membrane permeating agent.

When the compound is a protein or peptide, the complex is a fusion protein of the microbial membrane permeate and the compound (and optionally the linker). As a method for preparing the fusion protein, a known method such as a chemical synthesis method or a gene recombination method can be applied. In the case of the chemical synthesis method, a fusion protein is prepared using a commercially available peptide synthesizer, a peptide synthesis kit, or the like according to a known peptide synthesis method. In the case of the gene recombination method, the DNA encoding the membrane-permeable peptide in the microbial membrane-permeable agent and the DNA encoding the compound were directly linked or linked via a linker sequence and inserted into a known vector such as a plasmid. Later, by introducing the vector into the host cell, the complex can be expressed as a fusion protein in the host cell.

Since the complex of the present disclosure contains a microbial membrane permeating agent, it has the property of penetrating the cell membrane of a microorganism and can deliver the compound into the cell membrane. The complexes of the present disclosure can be applied in a variety of applications requiring delivery of compounds, such as drug delivery systems, into cell membranes.

≪Method of delivering compounds to microbial cells≫
In another aspect, the disclosure provides a method of delivering a compound to a microbial cell, comprising contacting the complex comprising the microbial membrane permeating agent of the present disclosure with the compound with the microbial cell. The microbial membrane penetrants, compounds and microorganisms are as described above.

In the present disclosure, "contact" refers to placing the cells under conditions of contact with the complex, which can be clearly understood by those skilled in the art.
The means for bringing the complex into contact with the microbial cells is not particularly limited, and those skilled in the art can carry out the procedure under appropriate conditions depending on the type of microorganism used, the type of compound contained in the complex, and the like. .. For example, as a means for bringing the complex into contact with the microbial cells, the following methods (1) to (3) can be mentioned.
(1) A method of culturing microbial cells in a medium to which a complex has been added.
(2) A method of adding or immersing microbial cells in a solution containing a complex.
(3) A method of laminating a complex on microbial cells.

Specific conditions for contacting the complex with the microbial cells, that is, the contact time, amount, number of times, temperature, pH, light, salt concentration, additive concentration, etc. between the microbial cells and the complex are known conditions. It may be appropriately designed based on.

The complex to be brought into contact may be one type or a combination of a plurality of types. For example, when examining the additive action, synergistic action, etc. of a plurality of compounds on microorganisms, the complex may be used in combination.

By the contact as described above, the compound contained in the complex is delivered into the cells of the microbial cells. Since the membrane-permeable peptides used in the present disclosure have high introduction efficiency and low toxicity to microorganisms, compound delivery to microorganisms according to the present disclosure has a great variety of uses.

≪Fungicide≫
In another aspect, the present disclosure provides a fungicide comprising the microbial membrane permeating agents of the present disclosure.
The disinfectants of the present disclosure include the microbial membrane permeating agents of the present disclosure.
The bactericidal agent of the present disclosure is a complex containing a microbial membrane permeating agent and a substance having a bactericidal action, whereby the above-mentioned bactericidal agent is highly efficient in the cells of a microorganism to be sterilized (particularly, a microorganism having drug resistance). A substance having a bactericidal action can be introduced.

The microorganism to be sterilized using the disinfectant of the present disclosure is not particularly limited, and examples thereof include the same types of microorganisms as those exemplified by the microbial membrane permeating agent of the present disclosure.

The use of the disinfectant of the present disclosure is not particularly limited, but can be used, for example, for preservatives, sterilization, sterilization, agriculture and horticulture, medical use, disinfection sterilization and the like.

In the present disclosure, the fungicide refers to any drug used to kill microorganisms, but the rate of fungicide is not limited and may be partial or complete inhibition.

≪Antibacterial agent≫
In another aspect, the disclosure provides an antibacterial agent comprising the microbial membrane permeating agent of the present disclosure.
The antibacterial agents of the present disclosure include the microbial membrane permeating agents of the present disclosure.
The antibacterial agent of the present disclosure is a complex containing a microbial membrane permeating agent and a substance having an antibacterial action, whereby the above-mentioned antibacterial agent is highly efficient in the cells of a microorganism to be antibacterial (particularly, a microorganism having drug resistance). A substance having an antibacterial action can be introduced.

The target microorganism to be antibacterial using the antibacterial agent of the present disclosure is not particularly limited, and examples thereof include the same types as the microorganisms exemplified by the microbial membrane permeating agent of the present disclosure.

The use of the antibacterial agent of the present disclosure is not particularly limited, but can be used, for example, for antiseptic, antibacterial, agricultural and horticultural, medical, disinfectant and sterilization.

In the present disclosure, the antibacterial agent refers to any drug used to inhibit the growth of microorganisms, but the rate of inhibiting the growth of microorganisms is not limited, and even partial inhibition is completely inhibited. It may be.

≪Labeling agent≫
In another aspect, the present disclosure provides a labeling agent comprising the microbial membrane permeating agents of the present disclosure.
The labeling agents of the present disclosure include the microbial membrane permeating agents of the present disclosure.
The labeling agent of the present disclosure is a complex containing a microbial membrane permeating agent and a substance capable of chemically interacting with and detecting a specific site in a target microorganism (hereinafter, also referred to as “labeling substance”). Is preferable. With the above configuration, it is possible to invade the cells of the target microorganism with high efficiency and label (label) a specific site in the target microorganism by luminescence observation, magnetism, optics, or the like.

A known method can be applied to the method for forming the complex.
The microbial membrane permeating agent and the labeling substance may be directly linked by a covalent bond or may be linked by an interaction such as a hydrogen bond. The labeling substance may be linked to the microbial membrane permeable agent, for example, by reacting with the amino group of another amino acid residue (eg, lysine, etc.) linked to the end of the membrane permeable peptide in the microbial membrane permeable agent.

The microorganism to be labeled with the labeling agent of the present disclosure is not particularly limited, and examples thereof include the same types of microorganisms as those exemplified by the microbial membrane permeating agent of the present disclosure.

The type of labeling substance is not particularly limited, but is specifically parallel to the base pairs of a double helix such as DNA or a fluorescent molecule having a reactive site capable of reacting with the specific site such as diazirine. Examples thereof include intercalator compounds (acridine and the like) to be inserted (intercalated).

The use of the labeling agent of the present disclosure is not particularly limited, but it can be used, for example, for biosensing, biolabeling, bioimaging, environmental inspection, and the like.

Examples will be illustrated below to illustrate the present disclosure, but these examples are provided solely for the purpose of explaining the present disclosure and limit or limit the scope of the invention disclosed in this application. It's not something to do.

[Example 1] Synthesis of membrane-permeable peptide and introduction of compound In this example, Dab9-K-FAM, Orn9-K-FAM, Dab4-K (-FAM) -Dab5 and (Dab4-K (-FAM) -Dab5, which have a fluorescent dye as a complex. DabFF) 3-K-FAM was synthesized.

(1) Synthesis of microbial membrane permeable agent In a solid-phase synthesis tube (polypropylene LibraTube main body tube 5 mL, Hypep Research Institute Co., Ltd.), Fmoc-NH-SAL resin (Watanabe Chemical Co., Ltd.) (64.1 mg, 0. 025 mmol) was immersed in N, N'-dimethylformamide (DMF) (Kishida Chemical Co., Ltd.) overnight and expanded. For the solid-phase synthesis tube, a solid-phase synthesis cap polypropylene LibraTube upper cap (Hypep Laboratory Co., Ltd.) was used. Piperidine (Kishida Chemical Co., Ltd.) (20% by mass in DMF, 2 mL) was added, and the mixture was vortexed for 1 minute to remove the solvent. Piperidine (20% by weight in DMF, 2 mL) was added and shaken at room temperature for 10 minutes to remove the solvent. Washed 5 times with DMF (2 mL). A small amount of resin was taken out, and it was confirmed that the resin was colored using TNBS Test Kit (Tokyo Chemical Industry Co., Ltd.). It was washed 3 times each with methylene chloride (2 mL) and DMF (2 mL). HBTU (Watanabe Chemical Co., _{Ltd.) 3.05 g, HOBt · H 2} O ( Watanabe Chemical Co., Ltd.) 1.25 g and premixed prepared condensing agent cocktail DMF 16mL (175μL), and N, N-diisopropylethylamine (DIEA) (Nakalitesk Co., Ltd.) and N-methyl-2-pyrrolidone (NMP) (Kishida Chemical Co., Ltd.) mixed solution (DIEA / NMP = 2.75 / 14.25 (v / v), 175 μL) , N-terminal amino acid (0.075 mmol) was added to dissolve and added to the resin. The solvent was removed by shaking at room temperature for 15 minutes. Washed 5 times with DMF (2 mL). A small amount of the resin was taken out, and it was confirmed using the TNBS Test Kit that the resin did not develop color. It was washed 3 times each with methylene chloride (Gordo Co., Ltd.) (2 mL) and DMF (2 mL). After that, the above operation was repeated according to the amino acid sequence to extend the peptide chain. For extension operations, Fmoc-Lys (Dde) -OH (Watanabe Chemical Co., Ltd.), Fmoc-Dab (Boc) -OH (Watanabe Chemical Co., Ltd.), Fmoc-Orn (Boc) -OH (Watanabe Chemical Co., Ltd.), Alternatively, Fmoc-Phe-OH (Watanabe Chemical Co., Ltd.) was used. For stirring, a dedicated shaking stirring system PetiSyzer PSP-5100 (Hypep Laboratory Co., Ltd.) was used.

Hydrazine monohydrate (Kishida Chemical Co., Ltd.) (2% by mass in DMF, 2 mL) was added to the stretched resin, and the mixture was shaken at room temperature for 10 minutes to remove the solvent. Washed 5 times with DMF (2 mL). A small amount of the resin was taken out, and it was confirmed that the resin was colored by using the TNBS Test Kit. It was washed 3 times each with methylene chloride (2 mL) and DMF (2 mL).

(2) Complex formation 5 (6) -HOBt · H ₂ O (19.1 mg) dissolved in DMF (2 mL) in 5 (6) -carboxyfluorescein (5-FAM) (Sigma-Aldrich) (28.2 mg, 0.075 mmol) , 0.125 mmol) was added and dissolved. N, N'-diisopropylcarbodiimide (DIC) (Fujifilm Wako Pure Chemical Industries, Ltd.) (23.4 μL, 0.15 mmol) and DIEA (25.8 μL, 0.15 mmol) were added thereto and added to the resin. .. It was shaded at room temperature and shaken overnight. The solvent was removed and washed 5 times with DMF (2 mL). Then, it was washed with methylene chloride (2 mL) and DMF (2 mL) three times each. Piperidine (20% by mass in DMF, 2 mL) was added and shaken at room temperature for 10 minutes to remove the solvent. Each was washed 5 times with DMF (2 mL), methylene chloride (2 mL) and MeOH (2 mL). It was dried in a desiccator for 20 minutes.

(3) Cutting out from the resin A deprotected cocktail (trifluoroacetic acid (TFA) (Kishida Chemical Co., Ltd.) 600 μL, triisopropylsilane (TIS) (Tokyo Chemical Industry Co., Ltd.) 15.8 μL, was added to the resin dried in a desiccator. 15.8 μL of water (prepared by mixing in advance) was added, and the mixture was gently shaken every 30 minutes at room temperature and allowed to stand for 90 minutes. The filtrate was collected in a 15 mL centrifuge tube. TFA (500 μL) was added to the synthesis tube, and the filtrate was collected in the centrifuge tube. This operation was repeated 3 times.

Diethyl ether (Kishida Chemical Co., Ltd.) (10 mL) was added to the centrifuge tube from which the filtrate was collected, and the mixture was sufficiently stirred. Centrifugation (4 ° C., 3500 × g, 5 min) was performed to remove the supernatant. This operation was repeated 3 times. It was allowed to stand in the draft for 10 minutes, dried, and then dried in a desiccator for 2 hours or more. The dried sample was dissolved in ion-exchanged water and lyophilized.

(4) Purification The peptide synthesized as described above was purified by HPLC. The operating conditions are as follows.
[Analytical HPLC]
Column: YMC-Triart C18 (250 x 4.6 mmφ)
Flow velocity: 1.0 mL / min
Measurement wavelength: 220 nm
Solvent: A: H ₂ O (containing 0.1% by mass TFA)
B: Acetonitrile (containing 0.1% by mass TFA)
Gradient condition: 0 min A: B = 90:10
20min A: B = 40:60
25min A: B = 90:10
30min A: B = 90:10
[Preparation HPLC]
Column: YMC-Actus Triart C18 (250 x 20.0 mmφ)
Flow velocity: 18.9 mL / min
Measurement wavelength: 220 nm
Solvent: A: H ₂ O (containing 0.1% by mass TFA)
B: Acetonitrile (containing 0.1% by mass TFA)
Gradient condition: 0 min A: B = 90:10
20min A: B = 40:60
25min A: B = 90:10
30min A: B = 90:10

By the above synthesis, the microbial membrane permeable agents having the molecular structures shown in FIGS. 1, 17 and 28 (Dab9-K-FAM, Orn9-K-FAM, Dab4-K (-FAM) -Dab5 and (DabFF) 3 -K-FAM) was obtained respectively. In FIG. 1, the left side portion is the structure of Dab9 and Orn9, respectively. In FIG. 1, the portion surrounded by a frame is a fluorescent FAM group introduced into the C-terminal lysine side chain (linker).

[Example 2] Introduction of complex into Escherichia coli-1
In this example, the introduction of the complex containing the membrane-permeable peptide prepared in Example 1 into Escherichia coli was investigated. Unless otherwise specified, all reagents used in the microbial experiment were commercially available special grade products or equivalent.
(1) Introduction of membrane-permeable peptide into Escherichia coli Escherichia coli K-12 strain (ATCC10798) was used for introduction. The cells cultured overnight were dispensed into 100 μL Eppen, centrifuged (7,500 × g, 10 min) and collected. Next, the recovered cells were suspended in phosphate buffered saline (PBS) (Thermo Fisher Scientific, pH 7.4), and centrifugation (7,500 × g, 5 min) was repeated 3 times and washed with PBS. .. To the cell pellet obtained after washing, 50 μL of 2 μM Dab9-K-FAM solution or Orn9-K-FAM solution was added, suspended, and then incubated at room temperature (23 ° C.) for 1 hour. After incubation, the cells were collected by centrifugation (7,500 × g, 5 min), and PBS washing was repeated 3 times in the same manner as described above. An appropriate amount of Trypan blue (Sigma-Aldrich, Trypan Blue solution, 0.4% by mass) diluted to 0.1% by mass was added to the finally obtained bacterial cell pellet, suspended, and then analyzed and observed. Was done.

However, since it was confirmed that Trypan Blue adsorbs non-specifically to the membrane in Gram-positive bacteria, the introduction experiment was performed only by staining with Dab9-K-FAM or Orn9-K-FAM.

(2) Observation with a fluorescence microscope The sample stained above was observed with a fluorescence microscope using 2 μL. The fluorescence microscope used was a BX53 upright microscope manufactured by OLYMPUS, and the light source device of the fluorescence microscope used U-HGLGPS manufactured by OLYMPUS. The excitation / absorption filter used in the microscopic observation was an OLYMPUS fluorescence mirror unit, and two types, U-FBW (for green fluorescence only) and U-FGW (for red fluorescence only), were used. The images were taken on a PC and the exposure times of the samples to be compared were the same.

Fluorescent Escherichia coli derived from FAM was observed with a fluorescence microscope, and the introduction of Dab9-K-FAM into the cells was directly observed. FIG. 2 shows a fluorescence microscope image (top) together with a photograph (bottom) showing the cells stained with trypan blue. In the figure, Negative control represents a system to which peptide is not added, and background fluorescence from the fungus is observed. Since they are all observed at the same exposure time, the magnitude of the fluorescence intensity corresponds to the amount of peptide having the fluorescence group FAM. From the results shown in FIG. 2, a known membrane permeable peptide KFFKFFKFFK _((KFF) 3 K: SEQ ID NO: 4) as compared to, DAB 9 is shown to have been significantly introduced into bacterial cells of E. coli.

Dap, Dab and Orn have 3 carbon, 2 carbon and 1 carbon shorter side chains than the natural amino acid lysine, respectively. However, the Dab and Orn dimers show more than twice the efficiency of introduction into E. coli as compared to the lysine dimer. It was unexpected that this slight difference in molecular structure between Dap, Dab and Orn and lysine would result in a large difference in membrane permeability.

(3) Quantitative evaluation of introduction into E. coli (flow cytometry)
BD's FACSAria ^TM II was used for analysis and sorting of the flow cytometer. The cells stained with Dab9-K-FAM or Orn9-K-FAM and Trypan Blue were adjusted to 1000 to 2000 events / s, and analyzed using BD's analysis software FACSDIVA. The number of samples analyzed in one sample was set to 30,000 events / s. Sorting was performed on the cells in which Dab9-K-FAM or Orn9-K-FAM was introduced, and 240 cells (single cells) were sorted on an LB agar medium plate. The sorted cells were cultured at an appropriate temperature, and the survival rate was calculated based on the number of colonies formed.

The results of flow cytometry are shown in FIGS. 3 and 4. In the figure, Escherichia coli not introduced into Dab9-K-FAM and Orn9-K-FAM was used as a control, and the introduction rate was evaluated. As a result, it was found that the complex containing the membrane-permeable peptide was introduced with high efficiency (introduction rate 100%).

[Example 3] Introduction of the complex into Escherichia coli-2
In this example, a complex containing a membrane-permeable peptide was prepared in the same manner as in Example 1, and introduction into Escherichia coli was examined in the same manner as in Example 2. Specifically, as the membrane-permeable peptide, Orn6 (SEQ ID NO: 7), Orn7 (SEQ ID NO: 8), Orn8 (SEQ ID NO: 9), Orn9 (SEQ ID NO: 3), Orn10 (SEQ ID NO: 10), Orn11 (SEQ ID NO: 10). 11) and Orn12 (SEQ ID NO: 12), Dab3 (Dab-Dab-Dab), Dab4 (SEQ ID NO: 20), Dab5 (SEQ ID NO: 21), Dab6 (SEQ ID NO: 13), Dab7 (SEQ ID NO: 14), Dab8 (SEQ ID NO: 14). No. 15), Dab9 (SEQ ID NO: 2), Dab10 (SEQ ID NO: 16), Dab11 (SEQ ID NO: 17) and Dab12 (SEQ ID NO: 18), Dab15 (SEQ ID NO: 24), Dab19 (SEQ ID NO: 25), and Dap9 (SEQ ID NO: 25). After each of No. 19) was prepared, a fluorescent FAM group was introduced via a lysine side chain linked to the C-terminal as a linker. Similarly, K (-FAM) -Dab9 (SEQ ID NO: 22) and Dab4-K (-FAM) -Dab5 (SEQ ID NO: 23) were used as membrane-permeable peptides to examine the introduction rate into Escherichia coli. ..

The introduction rate into E. coli was as follows.
Orn6-K-FAM: 4.00 ± 0.29%
Orn7-K-FAM: 16.90 ± 4.9%
Orn8-K-FAM: 97.20 ± 0.73%
Orn9-K-FAM: 99.88 ± 0.01%
Orn10-K-FAM: 99.93 ± 0.02%
Orn11-K-FAM: 99.94 ± 0.01%
Orn12-K-FAM: 99.89 ± 0.03%

Dab3-K-FAM: 0.41%
Dab4-K-FAM: 2.55 ± 0.20%
Dab5-K-FAM: 53.32 ± 0.06%
Dab6-K-FAM: 99.07 ± 0.68%
Dab7-K-FAM: 99.94 ± 0.02%
Dab8-K-FAM: 99.97 ± 0.01%
Dab9-K-FAM: 99.98 ± 0.01%
Dab10-K-FAM: 99.97 ± 0.01%
Dab11-K-FAM: 99.97 ± 0.01%
Dab12-K-FAM: 99.96 ± 0.02%
Dab15-K-FAM: 99.97 ± 0.02%
Dab19-K-FAM: 58.08 ± 0.47%

Dap9-K-FAM: 99.98 ± 0.01%
K (-FAM)-Dab9: 99.99%
Dab4-K (-FAM)-Dab5: 99.99%

As a result, it was found that the complex containing the membrane-permeable peptide was introduced with high efficiency.

[Example 4] Toxicity evaluation of the complex against Escherichia coli In this example, the toxicity of the membrane-permeable peptide to Escherichia coli was quantitatively evaluated (observation with a fluorescence microscope).

Escherichia coli showing fluorescence derived from FAM, that is, Escherichia coli 240 cells into which Dab9-K-FAM or Orn9-K-FAM was introduced was sorted into a medium, and after culturing for 1 day, the number of viable cells was formed. And counted the number of dead bacteria.

The subjects were 240 Escherichia coli randomly selected from 30,000 Escherichia coli used in each measurement, and after culturing for 1 day under the condition that Dab9-K-FAM or Orn9-K-FAM was not added, live colonies and live colonies. This is the result of counting dead colonies. The results are shown in Table 1 below.

From the results of the above direct observation, no significant increase in E. coli dead cells was observed due to the addition of Dab9-K-FAM or Orn9-K-FAM. Therefore, it was confirmed that the toxicity of the membrane-permeable peptides Dab9-K-FAM and Orn9-K-FAM is low.

[Example 5] Introduction of the complex into bacteria In this example, introduction into Yersinia bercovieri (gram-negative) and Bacillus megaterium (gram-positive) as bacterial species other than Escherichia coli. , Investigated by fluorescence microscopy.

Specifically, a composite containing a membrane-permeable peptide in a bacterial strain according to the same procedure as in Example 2 except that Yersinia bercobieri (NBRC105717) and Bacillus megaterium (inventor isolated strain; unregistered) were used. Body (Dab) ₉ K-FAM was introduced. The results are shown in FIGS. 5 and 6.

5 and 6 show a fluorescence microscope image (top) along with a photograph (bottom) showing the cells. In the figure, Negative control represents a system to which peptide is not added, and background fluorescence from the fungus is observed. Since they are all observed at the same exposure time, the magnitude of fluorescence intensity corresponds to the amount of peptide having the fluorescence group FAM. From the results of FIGS. 5 and 6, a known membrane permeable peptide (KFF) as compared to 3 _K, DAB 9 has been shown to significantly introduced into bacterial cells of gram-positive and gram-negative bacteria ..

[Example 6] Introduction of various complexes and evaluation of toxicity In this example, complexes containing the fluorescent dye FAM and the microbial membrane permeating agent of the present disclosure (Dab9-K-FAM and Orn9-K-FAM), In addition, a complex (KFF) ₃ K (SEQ ID NO: 4), K9 (SEQ ID NO: 5) and R9 (SEQ ID NO: 6) containing a fluorescent dye FAM and a known microbial membrane permeating agent was used. Incubation was performed at concentrations of 1, 2, 5, 10, and 20 μM, and the introduction rate and cytotoxicity of each complex were examined. The introduction rate was evaluated by the fluorescence derived from the fluorescent dye FAM in each complex. Cell toxicity was assessed using a flow cytometer by fluorescence of trypan blue-stained fungi. The results are shown in FIG.

As shown in FIG. 7, the complex (Dab9-K-FAM and Orn9-K-FAM) containing the fluorescent dye FAM and the microbial membrane permeating agent of the present disclosure contains the fluorescent dye FAM and a known microbial membrane permeating agent. Compared with the containing complex (KFF) ₃ K, K9 and R9, the introduction rate was almost 100% even at a low concentration.
In addition, as shown in the graph of the white symbol with "-D" in FIG. 7, all the complexes showed the ratio of dead cells of 2% or less. As described above, the complex (Dab9-K-FAM and Orn9-K-FAM) containing the fluorescent dye FAM and the microbial membrane permeating agent of the present disclosure has excellent cell-introducing properties and is toxic to the introduced cells. The effect of being very low has been shown.

[Example 7] Introduction of complex into bacteria In this example, gram-negative bacteria such as Yersinia bercovieri, Serratia grimesii, and Trouble Sierra guamensis are used as bacterial species other than Escherichia coli. (Trabulsiella guamensis), Enterobacter hormaechei, Proteus hauseri, Acinetobacter radioresistens, Pseudomonas fluorescens (Pseudomonas fluorescens) and Pseudomonas fluorescens (Pseudomonas fluorescens) The introduction of the complex Dab9-K-FAM was investigated by observing with a fluorescent microscope. The complex Dab9-K-FAM was introduced into the bacterial strain according to the same procedure as in Example 2 except that the bacterial species was changed. Further, in the same manner as in Example 2, a system in which no peptide was added (Negative control) and a system in which (KFF) 3K-FAM was introduced into a bacterial strain were also performed. The results of using each bacterium are shown in FIGS. 8 to 15.

The NBRC numbers of the above bacteria are as follows.
・ Yersinia Belcobieri (NBRC105717)
・ Serratia grimesis (NBRC13537)
・ Trouble Sierra Guamensis (NBRC103172)
・ Enterobacter Holmaechei (NBRC105718)
・ Proteus Hauseri (NBRC3851)
・ Acinetobacter Radio Resistance (NBRC102413)
・ Pseudomonas Fluorescence (NBRC14160)
・ Synechocystis PCC6803

8 to 15 show a fluorescence microscope image (top) together with a photograph (bottom) showing the bacterial cells.
In FIGS. 8 to 15, Negative control represents a system to which no peptide is added, and background fluorescence from the fungus is observed. Since they are all observed at the same exposure time, the magnitude of the fluorescence intensity corresponds to the amount of peptide having the fluorescence group FAM.
As shown in FIGS. 8-15, the complexes containing the microbial membrane permeable agents of the present disclosure are Gram-positive and Gram-negative bacteria compared to complexes containing known microbial membrane permeable agents or compared to Negative control. It was found that it was effectively introduced into the cells of.

[Example 8] Introduction of complex Dab4-K (-FAM) -Dab5 into bacteria In this example, the introduction of complex Dab4-K (-FAM) -Dab5 into Escherichia coli, which is a gram-negative bacterium, is fluorescent. Each was examined by microscopic observation. The complex was introduced into the bacterial strain according to the same procedure as in Example 2 except that the complex was changed to Dab4-K (-FAM) -Dab5. In addition, a system in which K (-FAM) -Dab9 was introduced into a bacterial strain was also performed according to the same procedure as in Example 2. The results are shown in FIG.

FIG. 16 shows a fluorescence microscope image (top) together with a photograph (bottom) showing the cells.
In FIG. 16, the background fluorescence from the fungus is observed, and all are observed at the same exposure time, so that the magnitude of the fluorescence intensity corresponds to the amount of the peptide having the fluorescence group FAM.
As shown in FIG. 16, the complex containing the microbial membrane permeating agent of the present disclosure (Dab4-K (-FAM) -Dab5 and K (-FAM) -Dab9) can be introduced into the cells of Escherichia coli. all right.

[Example 9] Introduction of complex (DabFF) ₃ K-FAM into bacteria In this example, Gram-negative bacteria, Acinetobacter radioresistens, Enterobacter hormaechei, and Escherichia coli. (Escherichia coil), Pantoea agglomerans, Proteus hauseri, Pseudomonas fluorescens, Serratia grimesii and trouble Sierra guamensis (Trabulsi) Was examined by observing with a fluorescent microscope. The complex (DabFF) ₃ K-FAM was introduced into the bacterial strain according to the same procedure as in Example 2 except that the bacterial species and the complex were changed. Further, in the same manner as in Example 2, a system in which no peptide was added (Negative control) and a system in which (KFF) 3K-FAM was introduced into a bacterial strain were also performed. The results of using each bacterium are shown in FIGS. 18 to 25.

The NBRC numbers of the above bacteria are as follows.
・ Acinetobacter Radio Resistance (NBRC102413)
・ Enterobacter Holmaechei (NBRC105718)
・ Escherichia coli (same species as Example 2)
・ Pantoea Agromelans (NBRC102470)
・ Proteus Hauseri (NBRC3851)
・ Pseudomonas Fluorescence (NBRC14160)
・ Serratia grimesis (NBRC13537)
・ Trouble Sierra Guamensis (NBRC103172)

18 to 25 show a fluorescence microscope image (top) together with a photograph (bottom) showing the cells.
18 to 25 show the background fluorescence from the fungus, and all are observed at the same exposure time, so that the magnitude of the fluorescence intensity corresponds to the amount of the peptide having the fluorescence group FAM.
As shown in FIGS. 18 to 25, it was found that the complex (DabFF) ₃ K-FAM containing the microbial membrane permeating agent of the present disclosure was introduced into each cell.

[Example 10] Evaluation of time- and concentration-dependent cytotoxicity of complex (Dab) ₉ K-FAM In this example, a complex containing the fluorescent dye FAM and the microbial membrane permeating agent of the present disclosure ((Dab) ) ₉ K-FAM) was used. Specifically, the complex is incubated with cells at concentrations of 0 μM, 2 μM and 20 μM for 0 hours, 1 hour and 24 hours, respectively, and contains a microbial membrane penetrant for normal human umbilical vein endothelial cells by microscopic observation. The cytotoxicity of the complex was evaluated. The results are shown in FIG.

[Example 11] Evaluation of time- and concentration-dependent cytotoxicity of complex (Orn) ₉ K-FAM In this example, a complex containing the fluorescent dye FAM and the microbial membrane permeating agent of the present disclosure ((Orn) ) ₉ K-FAM) was used. Specifically, the complex is incubated with cells at concentrations of 0 μM, 2 μM and 20 μM for 0 hours, 1 hour and 24 hours, respectively, and contains a microbial membrane penetrant for normal human umbilical vein endothelial cells by microscopic observation. The cytotoxicity of the complex was evaluated. The results are shown in FIG.

FIG. 26 shows micrographs showing the temporal and concentration-dependent cytotoxicity of the complex Dab9-K-FAM of the present disclosure to normal human umbilical vein endothelial cells.
FIG. 27 shows micrographs showing the temporal and concentration-dependent cytotoxicity of the complex Orn9-K-FAM of the present disclosure to normal human umbilical vein endothelial cells.
As shown in FIGS. 26 and 27, the complex containing the microbial membrane permeating agent of the present disclosure has low and limiting cytotoxicity to normal human umbilical vein endothelial cells in a time- and concentration-dependent manner. all right.

Claims (22)

A first membrane-permeable peptide containing 4 to 26 independently selected amino acid residues of formula (I) in succession, and a second membrane-permeable peptide containing amino acid residues of formula (II). A microbial membrane penetrant containing at least one of the sex peptides.

[In formulas (I) and (II), n is an independently integer of 1 to 3, and the amino acid residue may be either (R) -form or (S) -form. In formula (II), m is an integer of 1 to 5, and R ¹ and R ² independently represent hydrophobic substituents. In formulas (I) and (II), the site represented by * represents a binding site with an adjacent site. ] The first membrane-permeable peptide is (S) -2,5-diaminopentanoic acid (Orn), (R) -2,5-diaminopentanoic acid (Orn), (S) -2,4-diaminobutanoic acid. Consists of (Dab), (R) -2,4-diaminobutanoic acid (Dab), (S) -2,3-diaminopropionic acid (Dap) and (R) -2,3-diaminopropionic acid (Dap) The microbial membrane-penetrating agent according to claim 1, which comprises a membrane-permeable peptide consisting of 4 to 26 contiguous amino acid residues independently selected from the group. The microbial membrane-permeable agent according to claim 1 or 2, wherein the first membrane-permeable peptide comprises at least four consecutive Orn or Dab. The microbial membrane-permeable agent according to any one of claims 1 to 3, wherein the first membrane-permeable peptide comprises 5 to 26 consecutive Orn or Dab. The first membrane-permeable peptide is 5,6,7,8,9,10,11 or 12 consecutive (S) -Orn or 5,6,7,8,9,10,11 or 12 The microbial membrane permeating agent according to any one of claims 1 to 4, which comprises a series of (S) -Dab. The microbial membrane permeating agent according to any one of claims 1 to 5, wherein the first membrane-permeable peptide comprises 6 to 26 consecutive same amino acid residues. The first membrane-permeable peptide or the second membrane-permeable peptide contains one or several lysine (Lys) residues or arginine (Arg) residues in addition to the amino acid residues of the formula (I). The microbial membrane permeating agent according to any one of claims 1 to 6, which comprises. The first membrane-permeable peptide has a plurality of blocks consisting of the 4 to 26 amino acid residues of the formula (I) independently selected, and is located at least one of the plurality of blocks. The microbial membrane permeating agent according to any one of claims 1 to 7, further comprising one or two or more amino acid residues other than the amino acid residue of the formula (I) in succession. A claim that an amino acid residue other than the amino acid residue of the formula (I) contains a lysine (Lys) residue having a labeled molecule as a substituent or an arginine (Arg) residue having a labeled molecule as a substituent. 8. The microbial membrane permeating agent according to 8. Claims 1 to 9 wherein the microorganism is at least one selected from the group consisting of prokaryotes including eubacteria and archaea, and eukaryotes including microalgae, protists, fungi and slime molds. The microbial membrane permeating agent according to any one of the above. The microbial membrane penetrant according to any one of claims 1 to 10, wherein the microorganism is a bacterium belonging to the genus Enterobacter, Synechocystis, Pseudomonas, Acinetobacter or Vatilas. A complex containing the microbial membrane permeating agent according to any one of claims 1 to 11 and a compound. The compound is at least one selected from the group consisting of proteins, peptides, nucleic acids, peptide nucleic acids, inorganic substances, organic metal compounds, organic compounds, polymers, nanoparticles, molecular aggregates including micelles and liposomes, sugars and labeling substances. The complex according to claim 12, which comprises a species. The complex according to claim 12 or 13, wherein the microbial membrane permeating agent and the compound are linked directly or via a linker. The complex according to claim 14, wherein the linker consists of 1 to 10 consecutive amino acid residues. A method for delivering a compound to a microbial cell, which comprises contacting the complex containing the microbial membrane permeating agent according to any one of claims 1 to 11 with the microbial cell. The compound is selected from the group consisting of proteins, peptides, nucleic acids, peptide nucleic acids, inorganic substances, organic metal compounds, organic compounds, polymers, nanoparticles, sugars and labeling substances, and molecular aggregates containing micelles and liposomes. 16. The method of claim 16, comprising at least one of the above. 16 or 17 the microorganism is at least one selected from the group consisting of prokaryotes including eubacteria and archaea, and eukaryotes including microalgae, protists, fungi and slime molds. The method described in. The method according to any one of claims 16 to 18, wherein the microorganism is a bacterium belonging to the genus Enterobacter, Synechocystis, Pseudomonas, Acinetobacter or Bacillus. A bactericidal agent containing the microbial membrane permeating agent according to any one of claims 1 to 11. An antibacterial agent containing the microbial membrane permeating agent according to any one of claims 1 to 11. A labeling agent comprising the microbial membrane permeating agent according to any one of claims 1 to 11.