JP7480447B2 - Bacterial membrane permeabilizing agent, complex, method for delivering compound to bacterial cell using same, bactericide, antibacterial agent and labeling agent - Google Patents

Description

The present disclosure relates to bacterial membrane permeabilizing agents, complexes, methods of using same to deliver compounds to bacterial cells, bactericidal agents, antibacterial agents and labeling agents.

Membrane-permeable substances are effective molecular tools that enable the introduction of functional molecules and drugs into cells. They are used in various research fields related to biochemistry and pharmacology, and their importance and necessity are increasing. There has been active research into substances that can efficiently permeate the membranes of animal cells, and many reports have been published on peptides that permeate animal cell membranes (cell-penetrating peptides, CPPs). As an example of CPPs, a method has been reported in which arginine oligomers and lysine oligomers are bound to molecules as membrane-permeable peptides to allow the molecules to be taken up by cells (Patent Document 1).

On the other hand, there have been few research examples regarding membrane-permeable substances for prokaryotic cells such as microorganisms. The membrane structures of animal cells and microbial cells are significantly different. Specifically, animal cells only have a cell membrane, while microbial cells have a peptidoglycan layer on the outside of the cell membrane. Furthermore, gram-negative bacteria have an outer membrane on the outside of the peptidoglycan layer. Considering these structural differences and the multi-membrane structure of microorganisms, that is, the structure that prevents the permeation of substances, it is thought that there is no simple commonality between animal cell membrane-permeable substances and microbial membrane-permeable substances, and microbial membrane-permeable substances need to be developed independently from animal cell membrane-permeable substances.

For example, (RFF) ₃ (a nine amino acid peptide containing three repeats of arginine, phenylalanine, and phenylalanine), K9 (a peptide consisting of nine lysines), and R9 (a peptide consisting of nine arginines), which are known as cell-penetrating peptides for animal cells, have low introduction efficiencies for functional substances into microbial cells, with introduction efficiencies (actual measurements) for Escherichia coli being only 4.0%, 40.3%, and 68.2%, respectively. In addition, (KFF) _3K (a nine amino acid peptide containing three repeats of lysine, phenylalanine, and phenylalanine), which has been considered to have a good introduction rate for prokaryotic cells, also has an introduction efficiency (actual measurements) of about 12.8% for Escherichia coli.

There has also been a report on cell-penetrating peptides (CPPs) for E. coli (Non-Patent Document 1), but in this case the E. coli cells to be transfected are pretreated to make them more susceptible to uptake of the substance (competent cells), and then the substance bound to the cell-penetrating peptide is transfected.

JP 55-500053 A

K. Oikawa and 4 others, ACS Omega, American Chemical Society (USA), December 2018, Vol. 3, No. 12, pp. 16489-16499

The present disclosure therefore aims to provide a microbial membrane permeabilizing agent, a complex, a method for delivering a compound to a microbial cell using the same, a bactericide, an antibacterial agent, and a labeling agent that can permeate the cell membrane of a microorganism such as E. coli with high efficiency and low toxicity and introduce a target compound into the microbial cell.

As a result of intensive research to solve the above problems, the present inventors discovered that such a microbial membrane permeabilizing agent efficiently permeates the cell membrane of bacteria such as E. coli and has low cytotoxicity, leading to the completion of the present disclosure.

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

[1] A microbial membrane permeabilizing agent comprising at least one of a first membrane-permeable peptide containing 4 to 26 consecutive amino acid residues of formula (I), each independently selected from the other, and a second membrane-permeable peptide containing amino acid residues of formula (II).

[In formula (I) and formula (II), n is independently an integer of 1 to 3, and the amino acid residue may be either the (R) or (S) configuration. In formula (II), m is an integer of 1 to 5, and ^R1 and ^R2 each independently represent a hydrophobic substituent. In formula (I) and formula (II), the moiety represented by * represents a bonding site with an adjacent moiety.]
[2] The microbial membrane permeabilizing agent according to [1], wherein the first membrane-permeable peptide comprises a membrane-permeable peptide consisting of 4 to 26 consecutive amino acid residues independently selected from the group consisting of (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).
[3] The microbial membrane permeabilizing agent according to [1] or [2], wherein the first membrane-permeable peptide contains at least four consecutive Orn or Dab residues.
[4] The microbial membrane permeabilizing agent according to any one of [1] to [3], wherein the first membrane-permeable peptide consists of 5 to 26 consecutive Orn or Dab residues.
[5] The microbial membrane permeabilizing agent according to any one of [1] to [4], wherein the first membrane-permeable peptide consists of 5, 6, 7, 8, 9, 10, 11, or 12 consecutive (S)-Orn or 5, 6, 7, 8, 9, 10, 11, or 12 consecutive (S)-Dab.
[6] The microbial membrane permeabilizing agent according to any one of [1] to [5], wherein the first membrane-permeable peptide consists of 6 to 26 consecutive identical amino acid residues.
[7] The microbial membrane permeabilizing agent according to any one of [1] to [6], wherein the first membrane-permeable peptide or the second membrane-permeable peptide contains, in addition to the amino acid residues of formula (I), one or several lysine (Lys) residues or arginine (Arg) residues.
[8] The microbial membrane permeabilizing agent according to any one of [1] to [7], wherein the first membrane-permeable peptide has a plurality of blocks each consisting of 4 to 26 independently selected amino acid residues of formula (I), and contains one or more consecutive amino acid residues other than the amino acid residues of formula (I) at at least one position between the plurality of blocks.
[9] The microbial membrane permeabilizing agent according to [8], wherein the amino acid residue other than the amino acid residue of the formula (I) includes a lysine (Lys) residue having a labeling molecule as a substituent, or an arginine (Arg) residue having a labeling molecule as a substituent.
[10] The microbial membrane permeabilization agent according to any one of [1] to [9], wherein the microorganism is at least one species selected from the group consisting of prokaryotes including eubacteria and archaea, and eukaryotes including microalgae, protists, fungi, and slime molds.
[11] The microbial membrane permeabilization agent according to any one of [1] to [10], wherein the microorganism is a bacterium of the family Enterobacteraceae, the genus Synechocystis, the genus Pseudomonas, the genus Acinetobacter, or the genus Bacillus.
[12] A complex comprising the microbial membrane permeabilizing agent according to any one of [1] to [11] and a compound.
[13] The complex according to [12], wherein the compound comprises at least one selected from the group consisting of a protein, a peptide, a nucleic acid, a peptide nucleic acid, an inorganic substance, an organometallic compound, an organic compound, a polymer, a nanoparticle, a molecular assembly including a micelle and a liposome, a sugar, and a labeling substance.
[14] The complex according to [12] or [13], wherein the microbial membrane permeabilizing agent and the compound are linked directly or via a linker.
[15] The conjugate 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, comprising contacting a complex containing the microbial membrane permeabilizing agent according to any one of [1] to [11] and a compound with the microbial cell.
[17] The method according to [16], wherein the compound comprises at least one selected from the group consisting of a protein, a peptide, a nucleic acid, a peptide nucleic acid, an inorganic substance, an organometallic compound, an organic compound, a polymer, a nanoparticle, a sugar, a labeling substance, and a molecular assembly including a micelle and a liposome.
[18] The method according to [16] or [17], wherein the microorganism is at least one species selected from the group consisting of prokaryotes including eubacteria and archaea, and eukaryotes including microalgae, protists, fungi, and slime molds.
[19] The method according to any one of [16] to [18], wherein the microorganism is a bacterium of the family Enterobacteraceae, the genus Synechocystis, the genus Pseudomonas, the genus Acinetobacter, or the genus Bacillus.
[20] A bactericide comprising the microbial membrane permeabilization agent according to any one of [1] to [11].
[21] An antibacterial agent comprising the microbial membrane permeabilization agent according to any one of [1] to [11].
[22] A labeling agent comprising the microbial membrane permeabilization agent according to any one of [1] to [11].

The present disclosure provides a microbial membrane permeabilizing agent and complex that can permeate the cell membrane of a microorganism such as E. coli with high efficiency and low toxicity and introduce a target compound into the cell of the microorganism, a method for delivering a compound to a microbial cell using the same, a bactericide, an antibacterial agent, and a labeling agent.

FIG. 1 shows the structures of the complexes Dab9-K-FAM and Orn9-K-FAM of the present disclosure. 1 shows a fluorescence microscope image (top) and a photograph of bacterial cells (bottom) showing the membrane permeability of the complex of the present disclosure (Dab9-K-FAM) in Escherichia coli, a gram-negative bacterium. 1 shows the results of flow cytometry showing the log number of viable bacteria into which the complex of the present disclosure (Dab9-K-FAM) was introduced. 1 shows the results of flow cytometry showing the log number of viable bacteria into which the complex of the present disclosure (Dab9-K-FAM) was introduced. 1 shows 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 Yersinia bercovieri. Fluorescence microscopy images (top) and cell photographs (bottom) showing membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in the gram-positive bacterium Bacillus megaterium are shown. 1 is a graph showing the membrane permeability (solid symbols) and the percentage of dead cells (open symbols) of various membrane-permeable peptides. 1 shows 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 Yersinia bercovieri. 1 shows a fluorescence microscope image (top) and a photograph of the bacteria (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in the gram-negative bacterium Serratia grimesii. 1 shows a fluorescence microscope image (top) and a photograph of the bacteria (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in the Gram-negative bacterium Trabulsiella guamensis. 1 shows a fluorescence microscope image (top) and a photograph of the bacteria (bottom) showing membrane permeation of the complex (Dab9-K-FAM) of the present disclosure in the gram-negative bacterium Enterobacter hormaechei. 1 shows a fluorescence microscope image (top) and a photograph of the bacteria (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in the gram-negative bacterium Proteus hauseri. 1 shows a fluorescence microscope image (top) and a photograph of the bacteria (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in the gram-negative bacterium Acinetobacter radioresistens. 1 shows a fluorescence microscope image (top) and a photograph of the bacteria (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in the gram-negative bacterium Pseudomonas fluorescens. 1 shows a fluorescence microscope image (top) and a photograph of the bacteria (bottom) showing the membrane permeability of the complex (Dab9-K-FAM) of the present disclosure in the gram-negative bacterium Synechocystis PCC6803. Fluorescence microscopy images (top) and cell photographs (bottom) showing the membrane permeability of the complexes of the present disclosure (Dab9-K-FAM and Dab4-K(-FAM)-Dab5) in Escherichia coli, a gram-negative bacterium. FIG. 1 shows the structure of the complex of the present disclosure (Dab4-K(-FAM)-Dab5). 1 shows a fluorescence microscope image (top) and a photograph of the bacteria (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in the gram-negative bacterium Acinetobacter radioresistens. 1 shows a fluorescence microscope image (top) and a photograph of the bacteria (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in the gram-negative bacterium Enterobacter hormaechei. 1 shows a fluorescence microscope image (top) and a photograph of Escherichia coli (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in the gram-negative bacterium Escherichia coli. 1 shows a fluorescence microscope image (top) and a cell photograph (bottom) illustrating the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in the gram-negative bacterium Pantoea agglomerans. 1 shows a fluorescence microscope image (top) and a photograph of the bacteria (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in the gram-negative bacterium Proteus hauseri. 1 shows a fluorescence microscope image (top) and a photograph of the bacteria (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in the Gram-negative bacterium Pseudomonas fluorescens. 1 shows a fluorescence microscope image (top) and a photograph of the bacteria (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in the gram-negative bacterium Serratia grimesii. 1 shows a fluorescence microscope image (top) and a photograph of the bacteria (bottom) showing the membrane permeability of the complex (DabFF) ₃ K-FAM of the present disclosure in the Gram-negative bacterium Trabulsiella guamensis. 1 is a micrograph showing the time- and concentration-dependent cytotoxicity of the complex of the present disclosure, Dab9-K-FAM, against normal human umbilical vein endothelial cells. 1 is a micrograph showing the time- and concentration-dependent cytotoxicity of the complex Orn9-K-FAM of the present disclosure against normal human umbilical vein endothelial cells. FIG. 1 shows the structure of the complex ((DabFF)3-K-FAM) of the present disclosure.

This disclosure is explained in detail below.

In the present disclosure, a membrane-permeable peptide means a peptide capable of penetrating the cell membrane of a microorganism and entering the bacterial cell.
In this disclosure, "Dap" may be expressed as "Dpr" and "Dab" may be expressed 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 germicide in this disclosure includes not only what is generally called a germicide, but also what is called a sterilizer, disinfectant, and disinfectant.
In the present disclosure, the concept of antibacterial agent includes not only those generally called antibacterial agents, but also so-called bacteriostatic agents, bacteriostatic agents, antifungal agents, and the like.

In the present disclosure, the term "microbial membrane permeabilizing agent" refers to a permeabilizing agent that permeates the cell membrane of a microorganism and the peptidoglycan layer present on the cell membrane of the microorganism. The cell membrane of the microorganism also includes the spore coat and the spore cell membrane.

<Microbial membrane permeabilizer>
The present disclosure relates to a novel microbial membrane permeabilizing agent consisting of a membrane-permeable peptide, and uses thereof.
The microbial permeabilization agent of the present disclosure comprises at least one of a first membrane-permeable peptide comprising 4 to 26 consecutive amino acid residues of formula (I), each independently selected from the group consisting of 4 to 26 amino acid residues of formula (I), and a second membrane-permeable peptide comprising amino acid residues of formula (II).

In formula (I) and formula (II), n is independently an integer of 1 to 3, and the amino acid residue may be either the (R) or (S) configuration. In formula (II), m is an integer of 1 to 5, and ^R1 and ^R2 each independently represent a hydrophobic substituent. In formula (I) and formula (II), the moiety represented by * represents a bonding site with an adjacent moiety.

The microbial membrane permeabilizing agent of the present disclosure has the above-mentioned configuration, and is therefore capable of permeating the cell membrane of a microorganism such as E. coli with high efficiency and low toxicity, and of introducing a target compound into the cell of the microorganism.

The microbial membrane permeabilizing agent of the present disclosure may contain other components in addition to the membrane-permeable peptide, as long as the effect of the present disclosure is not inhibited. Examples of other components include stabilizers, preservatives, colorants, preservatives, thickeners, nutrients, antifungal agents, buffers, emulsifiers, organic solvents, etc.

[Membrane-permeable peptides]
The membrane-permeable peptide of the present disclosure is at least one of a first membrane-permeable peptide comprising 4 to 26 consecutive amino acid residues of formula (I) each independently selected, and a second membrane-permeable peptide comprising amino acid residues of formula (II).

(First membrane-permeable peptide)
The first membrane-permeable peptide comprises 4 to 26 consecutive, independently selected amino acid residues of formula (I).
In formula (I), n is independently an integer of 1 to 3, and the amino acid residues may be in either the (R) or (S) configuration.
In formula (I), the site represented by * represents a binding site to an adjacent site. The adjacent site represents a structure adjacent to an amino acid residue of formula (I), such as an adjacent amino acid residue, a terminal protecting group, a hydrogen atom at the N-terminus, a hydroxyl group at the C-terminus, or a linker described below.

Hereinafter, both the "microbial membrane-permeable peptide comprising 4 to 26 consecutive amino acid residues of formula (I) independently selected from each other" and the "membrane-permeable peptide comprising amino acid residues of formula (II)" are collectively referred to as the "microbial membrane-permeable peptide of the present disclosure."
Hereinafter, a "microbial membrane-permeable peptide comprising 4 to 26 consecutive amino acid residues independently selected from those of formula (I)" is also referred to as a "first microbial membrane-permeable peptide."
Hereinafter, the "membrane-permeable peptide comprising the amino acid residues of formula (II)" is also referred to as the "second microbial membrane-permeable peptide."

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

From the viewpoint of penetrating the cell membrane of a microorganism such as E. coli with higher efficiency and lower toxicity, the first membrane-permeable peptide preferably consists of 4 to 26 consecutive amino acid residues independently selected from the group consisting of (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).

In the present disclosure, when it is stated that a predetermined number of amino acid residues selected from a plurality of options are consecutive, the amino acid residues included in the consecutive amino acid residues may be the same or different from each other as long as they are selected from the plurality of options, and an example of this includes a predetermined number of consecutive identical amino acid residues.

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 are each independently (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-diaminopropionic acid (Dap);
X4 to X26 are each independently absent, (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-diaminopropionic acid (Dap).

The first membrane-permeable peptide is not particularly limited as long as it is a peptide containing 4 to 26 consecutive amino acid residues of formula (I) independently selected from each other.
Preferably, it is a peptide comprising 5 to 26 consecutive amino acid residues of formula (I) independently selected from each other,
More preferably, it is a peptide comprising 6 to 26 consecutive amino acid residues of formula (I) independently selected from each other;
More preferably, it is a peptide comprising 6 to 20 consecutive independently selected amino acid residues of formula (I).

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

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

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

From the viewpoint of further improving membrane permeability, the first membrane-permeable peptide is
Preferably, it comprises at least four consecutive Orn, Dab or Dap,
More preferably, it comprises at least four consecutive Orn or Dab;
More preferably, it comprises 5 to 26 consecutive Orn or Dab;
It is particularly preferred that it contains 6 to 26 consecutive Orns or Dabs.

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

More preferably, the first membrane-permeable peptide is composed of 6, 7, 8, 9, 10, 11, or 12 consecutive (S)-Orn, or 6, 7, 8, 9, 10, 11, or 12 consecutive (S)-Dab.

It is further preferred that the first membrane-permeable peptide consists of 8, 9, 10, 11, or 12 consecutive (S)-Orn, or 6, 7, 8, 9, 10, 11, or 12 consecutive (S)-Dab.

As examples, the amino acid sequences of the Dab9-mer (also referred to as Dab9 or (Dab)9) and the Orn9-mer (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)

The amino acid sequences of other first membrane-permeable peptides, namely 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)

In the specific example of the first membrane-permeable peptide, the stereoisomers of Orn, Dab, and Dap may all be in the (R) or (S) form, or may be a mixture of the (R) and (S) forms, but it is preferable that Orn, Dab, and Dap are all in the (S) form.

In one embodiment, the first membrane-permeable peptide may have a plurality of blocks consisting of the 4 to 26 independently selected amino acid residues of formula (I), and may include one or more consecutive amino acid residues other than the amino acid residues of formula (I) at at least one location between the plurality of blocks. In this case, the amino acid residues other than the amino acid residues of formula (I) preferably include a lysine (Lys) residue or an arginine (Arg) residue, and more preferably include a lysine (Lys) residue or an arginine (Arg) residue having a labeling molecule as a substituent. Examples of the labeling molecule include the same as the labeling molecules exemplified in the complex of the present disclosure described below.

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

In one embodiment, the first membrane-permeable peptide may be, for example, an amino acid sequence in which a single lysine (Lys) residue substituted with 5(6)-carboxyfluorescein (FAM), a labeling molecule, is present between a Dab tetramer and a Dab pentamer, as shown below.
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 amino acid residues of formula (II).
In formula (II), n is independently an integer from 1 to 3, and the amino acid residues may be in either the (R) or (S) configuration.
In formula (II), m is an integer of 1 to 5, and R ¹ and R ² each independently represent a hydrophobic substituent.
In formula (II), the site represented by * represents a binding site to an adjacent site. The adjacent site represents a structure adjacent to an amino acid residue of formula (II), such as an adjacent amino acid residue, a terminal protecting group, a hydrogen atom at the N-terminus, a hydroxyl group at the C-terminus, or a linker described below.

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

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

The hydrophobic substituent represented by ^R1 means that the amino acid residue containing NH and C═O at both ends of the carbon atom substituted by ^R1 (NH-C( ^R1 )-C(═O)) is derived from a hydrophobic amino acid.
The hydrophobic substituent represented by ^R2 means that the amino acid residue containing NH and C═O at both ends of the carbon atom substituted by ^R2 (NH-C( ^R2 )-C(═O)) is derived from a hydrophobic amino acid.

Hydrophobic amino acids refer to amino acids with a hydrophobic index (hi) of -2 or more. Specific examples of hydrophobic amino acids include glycine (hi = -0.4), tryptophan (hi = 0.9), methionine (hi = 1.9), proline (hi = -1.6), phenylalanine (hi = 2.8), alanine (hi = 1.8), valine (hi = 4.2), leucine (hi = 3.8), and isoleucine (hi = 4.5). Of the above, it is preferable to include phenylalanine as a hydrophobic amino acid.

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

In the above second membrane-permeable peptide, the stereoisomers may be such that Orn, Dab, and Dap are all in the (R) or (S) form, or may be a mixture of the (R) and (S) forms, but it is preferable that Orn, Dab, and Dap are all in the (S) form.

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

The second membrane-permeable peptide is, from the viewpoint of further improving membrane permeability,
Preferably, it comprises at least one Orn, Dab or Dap,
More preferably, it comprises at least two Orn or Dab;
More preferably, it contains 3 Dabs.

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

The microbial membrane permeabilizing agent of the present disclosure may contain at least one of the first membrane-permeable peptide and the second membrane-permeable peptide, or may contain both the first membrane-permeable peptide and the second membrane-permeable peptide. When the microbial membrane permeabilizing agent contains both the first membrane-permeable peptide and the second membrane-permeable peptide, the two may be connected via other amino acid residues described below.

The membrane-permeable peptide of the present disclosure may contain amino acid residues other than those of formula (I) and formula (II), so long as they do not affect microbial membrane permeability.
When the other amino acid residues are contained, the other amino acid residues are not particularly limited, but are preferably any 1 to 5 amino acid residues.
The other amino acid residues are preferably, for example, a lysine (Lys) residue or an arginine (Arg) residue.
The position of the other amino acid residue may be any of the terminals of the membrane-permeable peptide, between a plurality of amino acid residues of formula (I), between a plurality of amino acid residues of formula (II), and between an amino acid residue of formula (I) and an amino acid residue of formula (II). The other amino acid residue may have a labeling 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 and recombinant gene methods for introducing unnatural amino acids into polypeptides (e.g., Taira, H. et al., Biochem. Biophys. Res. Commun. 2008, 374, 304-308; Noren, C.J. et al., Science 244:182-188, 1989; Japanese Patent No. 4917044, etc.) can be used. Dab, Orn, and Dap are commercially available.

The membrane-permeable peptides disclosed herein can be easily synthesized with high yields using conventional solid-phase synthesis methods, and can also be mass-produced.

The microbial membrane permeabilizing agent according to the present disclosure can be linked to a compound to be introduced and brought into contact with a microorganism, thereby introducing the compound into the cell of the microorganism. In other words, the microbial membrane permeabilizing agent functions as a vector or carrier that carries various substances into the microbial cell. The compound to be introduced is a compound other than the microbial membrane permeabilizing 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 permeabilizing agent.

The "microorganisms" of interest include various types of microorganisms, including prokaryotes (e.g., eubacteria, archaea, etc.), eukaryotes (e.g., microalgae, protists, fungi, slime molds, etc.), and the like.
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, and the microbial membrane permeabilizing agent according to the present disclosure can be applied to both gram-negative and gram-positive bacteria.

Examples of bacteria include bacteria of the Enterobacteraceae family, the genus Synechocystis, the genus Pseudomonas, the genus Acinetobacter, or the genus Bacillus. Examples of bacteria of the Enterobacteraceae family include bacteria of the genus Yersinia, the genus Serratia, the genus Troublesiella, the genus Enterobacter, or the genus Proteus. Examples of bacteria of the Synechocystis genus include Synechocystis PCC6803, etc. Examples of bacteria of the Pseudomonas genus include Pseudomonas fluorescens, etc. Examples of bacteria of the Acinetobacter genus include Acinetobacter radioresistens, etc. Examples of bacteria of the Bacillus genus include Bacillus megaterium, etc.

The microbial membrane permeabilizing agent according to the present disclosure can permeate the cell membrane of microorganisms such as E. coli with high efficiency and low toxicity. Therefore, the microbial membrane permeabilizing agent according to the present disclosure can be used for basic research on the dynamics of microorganisms, the search for antibacterial substances, efficacy evaluation, antibacterial use, bactericidal use (sterilization), safety evaluation, medical use, environmental measurement, and other applications.

<Complex>
In another aspect, the present disclosure provides a complex comprising the microbial membrane permeabilizing agent of the present disclosure and a compound. The compound is not particularly limited as long as it is a compound that can be introduced into a microbial cell by permeating the microbial cell membrane.
The compound to be introduced into the bacterial cell preferably includes at least one selected from the group consisting of proteins, peptides, nucleic acids, peptide nucleic acids, inorganic substances, organometallic compounds, organic compounds, polymers, nanoparticles, sugars, labeling substances, and molecular assemblies including either micelles or liposomes. These compounds can be prepared by methods known in the art or obtained as commercially available products.

Examples of proteins include enzymes, receptors, antibodies, fluorescent proteins, cytoskeleton-forming proteins, and amyloid-forming proteins.
Examples of peptides include antigens, hormones, antimicrobial peptides, signal peptides, inhibitor peptides, redox-active peptides such as glutathione, and organelle-targeting peptides.
Examples of nucleic acids include DNA, RNA, hybrid nucleic acids, and aptamers.
Examples of peptide nucleic acids include peptide DNA and peptide RNA.
Inorganic compounds, organic compounds, organometallic compounds and nanoparticles include known compounds that can be introduced into cells.
Examples of the polymer include polyethylene glycol and dendrimers.
Examples of sugars include polysaccharides, disaccharides, monosaccharides, cyclodextrins, and the like.
An example of the labeling substance is 5(6)-carboxyfluorescein (FAM).
Examples of the molecular assembly include micelles, liposomes, etc. In the present disclosure, the term "molecular assembly" refers to an assembly of molecules such as lipids.

The method for linking the microbial membrane permeabilizing agent to the compound is not particularly limited, but examples thereof include (1) direct linkage (i.e., covalent bond); (2) linkage via a linker; (3) linkage by intermolecular interactions such as hydrogen bonds, hydrophobic interactions, electrostatic interactions, and coordinate bonds; and (4) linkage by antigen-antibody bonds, protein-ligand interactions (e.g., the interaction between avidin and biotin), DNA multiple helix formation, and the like, in which the intermolecular interactions act in combination.

In one embodiment, the method for linking the microbial membrane permeabilizing agent and the compound may be a method for linking the two by reacting an amino acid residue of the membrane-permeable peptide or an amino acid residue of the linker with a compound having a reactive functional group (an active ester group such as NHS ester, an epoxy group, an aldehyde group, etc.).

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

In one embodiment, the method for linking the microbial membrane permeabilizing agent and the compound may be a method in which the microbial membrane permeabilizing agent, which includes a membrane-permeable peptide in which a carboxylic acid group has been converted to an active ester by an activator or the like, is allowed to react with a compound having a reactive functional group to link the two.

When the microbial membrane permeabilizing agent and the compound are linked via a linker, a known linker sequence can be used as the linker sequence. For example, the linker sequence can be 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. Known amino acids can be used as the type of amino acid that forms the linker, and examples of such amino acids include lysine residues and arginine residues from the viewpoint of further maintaining the membrane permeability of the microbial membrane permeabilizing agent.

When the compound is a protein or peptide, the complex is a fusion protein of the microbial membrane permeabilizing agent and the compound (and optionally a linker). The fusion protein can be prepared by known methods such as chemical synthesis and genetic recombination. In the case of chemical synthesis, the fusion protein is prepared using a commercially available peptide synthesizer, peptide synthesis kit, or the like, according to a known peptide synthesis method. In the case of genetic recombination, the DNA encoding the membrane-permeable peptide in the microbial membrane permeabilizing agent and the DNA encoding the compound are linked directly or via a linker sequence, and then inserted into a known vector such as a plasmid, and the vector is introduced into a host cell, whereby the complex can be expressed as a fusion protein in the host cell.

The complex of the present disclosure contains a microbial membrane permeabilizing agent, and therefore has the property of permeating the cell membrane of a microorganism, and can deliver a compound into the cell membrane. The complex of the present disclosure can be applied to various applications that require the delivery of a compound into a cell membrane, such as a drug delivery system.

Method for delivering compounds to microbial cells
In another aspect, the present disclosure provides a method for delivering a compound to a microbial cell, comprising contacting the microbial cell with a complex comprising a microbial membrane permeabilizing agent of the present disclosure and a compound, wherein the microbial membrane permeabilizing agent, the compound, and the microorganism are as described above.

In the present disclosure, "contact" refers to placing the microbial cells and the complex under conditions in which they come into contact, as will be clearly understood by those skilled in the art.
The means for contacting the complex with the microbial cells is not particularly limited, and a person skilled in the art can carry out the contact under appropriate conditions depending on the type of microorganism used, the type of compound contained in the complex, etc. For example, the means for contacting the complex with the microbial cells include the following techniques (1) to (3).
(1) A technique in which microbial cells are cultured in a medium to which the complex has been added.
(2) A method in which microbial cells are added to or immersed in a solution containing the complex.
(3) A method of layering the complex on microbial cells.

The specific conditions for contacting the complex with the microbial cells, i.e., the contact time, amount, number of times, temperature, pH, light, salt concentration, additive concentration, etc., between the microbial cells and the complex may be appropriately designed based on known conditions.

The complex to be contacted may be one type, or multiple types may be combined. For example, when examining additive or synergistic effects of multiple compounds on microorganisms, a combination of complexes may be used.

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

<Disinfectant>
In another aspect, the present disclosure provides a disinfectant comprising the microbial membrane permeabilization agent of the present disclosure.
The disinfectant of the present disclosure includes the microbial membrane permeabilizer of the present disclosure.
The bactericide of the present disclosure is a complex containing a microbial membrane permeabilizing agent and a substance having bactericidal activity, which allows the substance having bactericidal activity to be introduced with high efficiency into the cells of the microorganisms to be sterilized (especially microorganisms having drug resistance).

The microorganisms to be sterilized using the germicide of the present disclosure are not particularly limited, but examples include the same types of microorganisms as those exemplified as the microbial membrane permeabilizer of the present disclosure.

The uses of the fungicide disclosed herein are not particularly limited, but can be used, for example, for preservation, disinfection, sterilization, agricultural and horticultural purposes, medical purposes, disinfection, and sterilization, etc.

In this disclosure, a disinfectant refers to any agent used to kill microorganisms, but the percentage of killing is not limited, and may be partial or complete inhibition.

<Antibacterial agents>
In another aspect, the present disclosure provides an antimicrobial agent comprising a microbial membrane permeabilization agent of the present disclosure.
The antimicrobial agents of the present disclosure include the microbial membrane permeabilizing agents of the present disclosure.
The antibacterial agent of the present disclosure is a complex containing a microbial membrane permeabilizing agent and a substance having antibacterial activity, which allows the substance having antibacterial activity to be introduced with high efficiency into the cells of the microorganism to be treated with antibacterial agents (especially drug-resistant microorganisms).

The microorganisms to be treated with the antibacterial agent of the present disclosure are not particularly limited, but may be, for example, the same types of microorganisms as those exemplified as the microbial membrane permeabilizing agent of the present disclosure.

The uses of the antibacterial agent disclosed herein are not particularly limited, but can be used, for example, for preservation, antibacterial, agricultural and horticultural purposes, medical purposes, disinfection, sterilization, etc.

In this disclosure, an antimicrobial agent refers to any agent used to inhibit the growth of microorganisms, but the percentage of inhibition of microbial growth is not limited, and may be partial or complete.

Labeling Agents
In another aspect, the present disclosure provides a labeling agent comprising a microbial membrane permeabilization agent of the present disclosure.
The labeling agents of the present disclosure include the microbial membrane permeabilizing agents of the present disclosure.
The labeling agent of the present disclosure is preferably a complex containing a microbial membrane permeabilizing agent and a substance capable of chemically interacting with and detecting a specific site in a target microorganism (hereinafter also referred to as a "labeling substance"). By adopting the above-mentioned configuration, it is possible to highly efficiently invade the cells of the target microorganism and label (label) the specific site in the target microorganism by luminescence observation, magnetism, optics, or the like.

The complex can be formed by a known method.
The microbial membrane permeabilizing 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. For example, the labeling substance may be linked to the microbial membrane permeabilizing agent by reacting with an amino group of another amino acid residue (e.g., lysine, etc.) linked to the terminus of the membrane-permeable peptide in the microbial membrane permeabilizing agent.

The microorganisms to be labeled using the labeling agent of the present disclosure are not particularly limited, but may be, for example, the same types of microorganisms as those exemplified as the microbial membrane permeabilizing agent of the present disclosure.

The type of labeling substance is not particularly limited, but examples include fluorescent molecules having a reactive site that can react with the specific site, such as diazirine, and intercalator compounds (acridine, etc.) that specifically insert (intercalate) in parallel between base pairs of a double helix such as DNA.

The uses of the labeling agent of the present disclosure are not particularly limited, but can be used for, for example, biosensing, biolabeling, bioimaging, environmental testing, etc.

The following examples are provided to illustrate the present disclosure, but they are provided merely to illustrate the present disclosure and are not intended to limit or restrict the scope of the invention disclosed in this application.

[Example 1] Synthesis of membrane-permeable peptides and introduction of compounds In this example, Dab9-K-FAM, Orn9-K-FAM, Dab4-K(-FAM)-Dab5, and (DabFF)3-K-FAM, which have fluorescent dyes as conjugates, were synthesized.

(1) Synthesis of microbial membrane permeabilizer In a solid-phase synthesis tube (polypropylene LibraTube main tube 5 mL, Hi-Pep Laboratory Co., Ltd.), Fmoc-NH-SAL resin (Watanabe Chemical Co., Ltd.) (64.1 mg, 0.025 mmol) was immersed overnight in N,N'-dimethylformamide (DMF) (Kishida Chemical Co., Ltd.) and expanded. A solid-phase synthesis cap polypropylene LibraTube upper cap (Hi-Pep Laboratory Co., Ltd.) was used for the solid-phase synthesis tube. Piperidine (Kishida Chemical Co., Ltd.) (20% by mass in DMF, 2 mL) was added, and the mixture was stirred with a vortex for 1 minute to remove the solvent. Piperidine (20% by mass in DMF, 2 mL) was added, and the mixture was shaken at room temperature for 10 minutes to remove the solvent. The mixture was washed five times with DMF (2 mL). A small amount of the resin was taken out, and the resin was confirmed to be colored using a TNBS Test Kit (Tokyo Chemical Industry Co., Ltd.). The resin was washed three times with methylene chloride (2 mL) and DMF (2 mL). A condensation agent cocktail (175 μL) was prepared by mixing 3.05 g of HBTU (Watanabe Chemical Co., Ltd.), 1.25 g of HOBt.H ₂ O (Watanabe Chemical Co., Ltd.), and 16 mL of DMF, and a mixture of N,N-diisopropylethylamine (DIEA) (Nacalai Tesque Co., Ltd.) and N-methyl-2-pyrrolidone (NMP) (Kishida Chemical Co., Ltd.) (DIEA/NMP=2.75/14.25 (v/v), 175 μL) was added to the N-terminal amino acid (0.075 mmol) and dissolved, and added to the resin. The resin was shaken at room temperature for 15 minutes to remove the solvent. The resin was washed five times with DMF (2 mL). A small amount of the resin was taken out, and it was confirmed that the resin did not color using the TNBS Test Kit. The mixture was washed three times with methylene chloride (GODO Co., Ltd.) (2 mL) and DMF (2 mL). The above procedure was then repeated according to the amino acid sequence to elongate the peptide chain. For the elongation procedure, Fmoc-Lys(Dde)-OH (Watanabe Chemical Co., Ltd.), Fmoc-Dab(Boc)-OH (Watanabe Chemical Co., Ltd.), Fmoc-Orn(Boc)-OH (Watanabe Chemical Co., Ltd.), or Fmoc-Phe-OH (Watanabe Chemical Co., Ltd.) was used. For stirring, a shaking and stirring system PetiSyzer PSP-5100 (Hi-Pep Research Institute Co., Ltd.) was used.

After the extension was completed, hydrazine monohydrate (Kishida Chemical Co., Ltd.) (2% by weight in DMF, 2 mL) was added to the resin, and the resin was shaken at room temperature for 10 minutes to remove the solvent. The resin was washed five times with DMF (2 mL). A small amount of the resin was removed, and the resin was confirmed to be colored using a TNBS Test Kit. The resin was washed three times each with methylene chloride (2 mL) and DMF (2 mL).

(2) Formation of Complex 5(6)-Carboxyfluorescein (5-FAM) (Sigma-Aldrich) (28.2 mg, 0.075 mmol) was dissolved in DMF (2 mL) and HOBt.H ₂ O (19.1 mg, 0.125 mmol) was added to dissolve the compound. 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 to the resin. The resin was shaken overnight at room temperature in the dark. The solvent was removed and the resin was washed five times with DMF (2 mL). The resin was then washed three times each with methylene chloride (2 mL) and DMF (2 mL). Piperidine (20% by mass in DMF, 2 mL) was added and the resin was shaken at room temperature for 10 minutes to remove the solvent. The mixture was washed five times each with DMF (2 mL), methylene chloride (2 mL), and MeOH (2 mL), and then dried in a desiccator for 20 minutes.

(3) Cutting out from resin A deprotection cocktail (pre-prepared by mixing 600 μL of trifluoroacetic acid (TFA) (Kishida Chemical Co., Ltd.), 15.8 μL of triisopropylsilane (TIS) (Tokyo Chemical Industry Co., Ltd.), and 15.8 μL of water) was added to the resin dried in a desiccator, 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 same centrifuge tube. This operation was repeated three 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 thoroughly stirred. The mixture was centrifuged (4°C, 3500 x g, 5 min) and the supernatant was removed. This procedure was repeated three times. The mixture was left to stand in a draft for 10 minutes and then dried in a desiccator for at least 2 hours. The dried sample was dissolved in ion-exchanged water and freeze-dried.

(4) Purification The peptides synthesized as described above were purified by HPLC under the following operating conditions.
Analytical HPLC
Column: YMC-Triart C18 (250 x 4.6 mmφ)
Flow rate: 1.0 mL / min
Measurement wavelength: 220 nm
Solvent: A: _H2O (containing 0.1% by weight TFA)
B: Acetonitrile (containing 0.1% by mass TFA)
Gradient conditions: 0 min A:B = 90:10
20min A:B=40:60
25min A:B=90:10
30min A:B=90:10
[Preparative HPLC]
Column: YMC-Actus Triart C18 (250 x 20.0 mmφ)
Flow rate: 18.9 mL / min
Measurement wavelength: 220 nm
Solvent: A: _H2O (containing 0.1% by weight TFA)
B: Acetonitrile (containing 0.1% by mass TFA)
Gradient conditions: 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, microbial membrane permeabilizing agents (Dab9-K-FAM, Orn9-K-FAM, Dab4-K(-FAM)-Dab5 and (DabFF)3-K-FAM) with the molecular structures shown in Figures 1, 17 and 28, respectively, were obtained. In Figure 1, the left side parts are the structures of Dab9 and Orn9, respectively. In Figure 1, the boxed parts are the fluorescent FAM group introduced into the C-terminal lysine side chain (linker).

[Example 2] Introduction of the complex into E. coli-1
In this example, we investigated the introduction into Escherichia coli of the complex containing the membrane-permeable peptide prepared in Example 1. Unless otherwise specified, all reagents used in the microbial experiments were commercially available premium grade products or equivalent.
(1) Introduction of membrane-permeable peptides into Escherichia coli Escherichia coli K-12 strain (ATCC10798) was used for the introduction. Bacteria cultured overnight were dispensed into 100 μL Eppendorf tubes and collected by centrifugation (7,500×g, 10 min). The collected bacteria were then suspended in phosphate-buffered saline (PBS) (Thermo Fisher Scientific, pH 7.4), centrifuged three times (7,500×g, 5 min), and washed with PBS. 50 μL of 2 μM Dab9-K-FAM solution or Orn9-K-FAM solution was added to the bacterial pellet obtained after washing, suspended, and then incubated at room temperature (23° C.) for 1 hour. After incubation, the bacteria were collected by centrifugation (7,500×g, 5 min), and washed with PBS three times in the same manner as above. Finally, 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 obtained bacterial pellet, and the mixture was suspended, followed by analysis and observation.

However, since it has been confirmed that Trypan Blue nonspecifically adsorbs to the membrane of Gram-positive bacteria, the introduction experiment was performed using only Dab9-K-FAM or Orn9-K-FAM staining.

(2) Fluorescence Microscope Observation Observation was performed using 2 μL of the stained sample using a fluorescence microscope. The fluorescence microscope used was an upright BX53 microscope manufactured by Olympus Corporation, and the light source device of the fluorescence microscope was an U-HGLGPS manufactured by Olympus Corporation. The excitation/absorption filters used in the microscope observation were two types of fluorescent mirror units manufactured by Olympus Corporation, U-FBW (for green fluorescence only) and U-FGW (for red fluorescence only). Images were taken on a PC, and the exposure times of the samples to be compared were aligned.

E. coli emitting fluorescence derived from FAM was observed under a fluorescent microscope, and the introduction of Dab9-K-FAM into the cells was directly observed. Figure 2 shows a photograph (bottom) of trypan blue-stained cells together with a fluorescent microscope image (top). In the figure, the negative control represents a system in which no peptide was added, and background fluorescence from the cells is observed. Since all observations were performed with the same exposure time, the magnitude of the fluorescence intensity corresponds to the amount of peptide having the fluorophore FAM. The results in Figure 2 show that Dab9 was significantly introduced into E. coli cells, compared with the known membrane-permeable peptide KFFKFFKFFK ((KFF) ₃ K: SEQ ID NO: 4).

Dap, Dab and Orn have side chains that are three, two and one carbon shorter than the natural amino acid lysine, respectively. However, the Dab and Orn nonamers are more than twice as efficient at introducing into E. coli as the lysine nonamer. It was unexpected that this slight difference in molecular structure between Dap, Dab and Orn and lysine would result in such a large difference in membrane permeability.

(3) Quantitative evaluation of transfection into E. coli (flow cytometry)
Analysis and sorting of the flow cytometer were performed using BD's FACSAria ^TM II. The cells stained with Dab9-K-FAM or Orn9-K-FAM and Trypan Blue were adjusted to 1000-2000 events/s, and analyzed using BD's analysis software FACSDIVA. The number of samples analyzed per 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 onto an LB agar medium plate. The sorted cells were cultured at an appropriate temperature, and the survival/death ratio was calculated based on the number of colonies formed.

The results of flow cytometry are shown in Figures 3 and 4. In the figures, E. coli that had not been transfected with Dab9-K-FAM or Orn9-K-FAM was used as a control, and the transfection rate was evaluated. As a result, it was found that the complex containing the membrane-permeable peptide was transfected with high efficiency (transfection rate of 100%).

[Example 3] Introduction of the complex into E. coli -2
In this example, a complex containing a membrane-permeable peptide was prepared in the same manner as in Example 1, and its introduction into E. coli was examined in the same manner as in Example 2. Specifically, 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:11), 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:15), Dab9 (SEQ ID NO:2), Dab10 (SEQ ID NO:16), Dab11 (SEQ ID NO:17), Dab12 (SEQ ID NO:18), Dab15 (SEQ ID NO:24), Dab19 (SEQ ID NO:25), and Dap9 (SEQ ID NO:19) were prepared as membrane-permeable peptides, and then a fluorescent FAM group was introduced into each of them via a lysine side chain linked to the C-terminus as a linker. Similarly, the introduction rates into E. coli were examined using K(-FAM)-Dab9 (SEQ ID NO: 22) and Dab4-K(-FAM)-Dab5 (SEQ ID NO: 23) as membrane-permeable peptides.

The introduction rates into E. coli were 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%

The results showed that the complex containing the membrane-permeable peptide was introduced with high efficiency.

Example 4: Evaluation of toxicity of complex against E. coli In this example, the toxicity of the membrane-permeable peptide against E. coli was quantitatively evaluated (observation with a fluorescence microscope).

E. coli showing fluorescence derived from FAM, i.e., E. coli 240 cells transfected with Dab9-K-FAM or Orn9-K-FAM, were sorted into medium, and after one day of culture, the number of live and dead bacteria in the colonies formed were counted.

The E. coli used in each measurement was randomly selected from 30,000 cells, and after culturing for one day without adding Dab9-K-FAM or Orn9-K-FAM, the number of live and dead colonies was counted. The results are shown in Table 1 below.

These direct observation results showed that the addition of Dab9-K-FAM or Orn9-K-FAM did not result in a significant increase in dead E. coli cells. This 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 of the complex into bacterial species other than E. coli, Yersinia bercovieri (gram-negative) and Bacillus megaterium (gram-positive), was examined by observation under a fluorescent microscope.

Specifically, except that Yersinia bercovieri (NBRC105717) and Bacillus megaterium (strain isolated by the inventor; unregistered) were used, the complex (Dab) _9K -FAM containing the membrane-permeable peptide was introduced into the bacterial strains according to the same procedure as in Example 2. The results are shown in Figures 5 and 6.

Figures 5 and 6 show photos of bacteria (bottom) and fluorescence microscopy images (top). In the figures, the negative control represents a system to which no peptide was added, and shows background fluorescence from bacteria. Since all observations were performed with the same exposure time, the magnitude of the fluorescence intensity corresponds to the amount of peptide having the fluorophore FAM. The results in Figures 5 and 6 show that Dab9 was significantly introduced into the cells of Gram-positive and Gram-negative bacteria, compared to the known membrane-permeable peptide (KFF) ₃ K.

[Example 6] Evaluation of introduction and toxicity of various complexes In this example, complexes (Dab9-K-FAM and Orn9-K-FAM) containing the fluorescent dye FAM and the microbial membrane permeabilizing agent of the present disclosure, and complexes (KFF) ₃ containing the fluorescent dye FAM and a known microbial membrane permeabilizing agent were used. K (SEQ ID NO: 4), K9 (SEQ ID NO: 5) and R9 (SEQ ID NO: 6) were used. The complexes were incubated 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. The toxicity of the cells was evaluated using a flow cytometer by the fluorescence of bacteria stained with trypan blue. The results are shown in FIG. 7.

As shown in FIG. 7, the complexes containing the fluorescent dye FAM and the microbial membrane permeabilizing agent of the present disclosure (Dab9-K-FAM and Orn9-K-FAM) showed an introduction rate of nearly 100% even at low concentrations, compared to the complexes containing the fluorescent dye FAM and the known microbial membrane permeabilizing agent (KFF) ₃ K, K9, and R9.
In addition, as shown in the graph of the open symbol "-D" in Figure 7, all the complexes showed a rate of dead cells of 2% or less. Thus, it has been demonstrated that the complexes containing the fluorescent dye FAM and the microbial membrane permeabilizing agent of the present disclosure (Dab9-K-FAM and Orn9-K-FAM) have excellent cell introduction properties and are very low in toxicity to the introduced cells.

[Example 7] Introduction of the complex into bacteria In this example, the introduction of the complex Dab9-K-FAM was examined by fluorescence microscopy using the gram-negative bacteria Yersinia bercovieri, Serratia grimesii, Trabulsiella guamensis, Enterobacter hormaechei, Proteus hauseri, Acinetobacter radioresistens, Pseudomonas fluorescens and Synechocystis PCC6803 as bacterial species other than E. coli. The complex Dab9-K-FAM was introduced into the bacterial strains according to the same procedure as in Example 2, except that the bacterial species was changed. In addition, a system in which no peptide was added (negative control) and a system in which (KFF)3K-FAM was introduced into the bacterial strain were also performed in the same manner as in Example 2. The results using each type of bacteria are shown in Figures 8 to 15.

The NBRC numbers of the above bacteria are as follows:
- Yersinia berthobieri (NBRC105717)
・Serratia grimecii (NBRC13537)
-Troublesiella guamensis (NBRC103172)
・Enterobacter hormaechei (NBRC105718)
・Proteus hauseli (NBRC3851)
・Acinetobacter radioresistens (NBRC102413)
- Pseudomonas fluorescens (NBRC14160)
・Synechocystis PCC6803

8 to 15 show photographs (bottom) of the cells and fluorescent microscopic images (top).
In Figures 8 to 15, the negative control represents a system in which no peptide was added, and background fluorescence from the bacteria is observed. Since all observations were performed with the same exposure time, the magnitude of the fluorescence intensity corresponds to the amount of peptide having the fluorophore FAM.
As shown in Figures 8 to 15, it was found that the complex containing the microbial membrane permeabilizing agent of the present disclosure was effectively introduced into the cells of Gram-positive and Gram-negative bacteria compared to the complex containing the known microbial membrane permeabilizing agent or compared to the negative control.

[Example 8] Introduction of the complex Dab4-K(-FAM)-Dab5 into bacteria In this example, the introduction of the complex Dab4-K(-FAM)-Dab5 into the gram-negative bacterium Escherichia coli was examined by fluorescence microscopy. 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 the bacterial strain was also performed according to the same procedure as in Example 2. The results are shown in FIG. 16.

FIG. 16 shows a photograph (bottom) of the cells and a fluorescent microscope image (top).
FIG. 16 shows background fluorescence from bacteria, and since all observations were made with the same exposure time, the magnitude of the fluorescence intensity corresponds to the amount of peptide having the fluorophore FAM.
As shown in FIG. 16, it was found that complexes containing the microbial membrane permeabilizing agent of the present disclosure (Dab4-K(-FAM)-Dab5 and K(-FAM)-Dab9) were introduced into the cells of E. coli.

[Example 9] Introduction of the complex (DabFF) _3K -FAM into bacteria In this example, introduction into each of the gram-negative bacteria Acinetobacter radioresistens, Enterobacter hormaechei, Escherichia coil, Pantoea agglomerans, Proteus hauseri, Pseudomonas fluorescens, Serratia grimesii, and Trabulsiella guamensis was examined by fluorescence microscopy. The complex (DabFF) 3K-FAM was introduced into the bacterial strains according to the same procedure as in Example 2, except that the bacterial species and complex were changed. In addition, a system without peptide (negative control) and a system in which (KFF) _3K -FAM was introduced into the bacterial strains were also performed in the same manner as in Example 2. The results using each of the bacteria are shown in FIGS.

The NBRC numbers of the above bacteria are as follows:
・Acinetobacter radioresistens (NBRC102413)
・Enterobacter hormaechei (NBRC105718)
- Escherichia coli (same species as in Example 2)
・Pantoea agglomerans (NBRC102470)
・Proteus hauseli (NBRC3851)
- Pseudomonas fluorescens (NBRC14160)
・Serratia grimecii (NBRC13537)
-Troublesiella guamensis (NBRC103172)

18 to 25 show photographs (bottom) of the cells and fluorescent microscopic images (top).
18 to 25 show background fluorescence from bacteria, and since all observations were made with the same exposure time, the magnitude of the fluorescence intensity corresponds to the amount of peptide having the fluorophore FAM.
As shown in FIG. 18 to FIG. 25, it was found that the complex (DabFF) ₃ K-FAM containing the microbial membrane permeabilizing agent of the present disclosure was introduced into each bacterial cell.

[Example 10] Evaluation of time-dependent and concentration-dependent cytotoxicity of the complex (Dab) ₉ K-FAM In this example, a complex ((Dab) ₉ K-FAM) containing the fluorescent dye FAM and the microbial membrane permeabilizing agent of the present disclosure was used. Specifically, the complex was incubated with cells at concentrations of 0 μM, 2 μM, and 20 μM for 0 hours, 1 hour, and 24 hours, respectively, and the cytotoxicity of the complex containing the microbial membrane permeabilizing agent against normal human umbilical vein endothelial cells was evaluated by microscopic observation. The results are shown in FIG. 26.

Example 11 Evaluation of time-dependent and concentration-dependent cytotoxicity of the complex (Orn) ₉ K-FAM In this example, a complex ((Orn) ₉ K-FAM) containing the fluorescent dye FAM and the microbial membrane permeabilizing agent of the present disclosure was used. Specifically, the complex was incubated with cells at concentrations of 0 μM, 2 μM, and 20 μM for 0 hours, 1 hour, and 24 hours, respectively, and the cytotoxicity of the complex containing the microbial membrane permeabilizing agent against normal human umbilical vein endothelial cells was evaluated by microscopic observation. The results are shown in FIG. 27.

FIG. 26 shows micrographs illustrating the time- and concentration-dependent cytotoxicity of the complex of the present disclosure, Dab9-K-FAM, against normal human umbilical vein endothelial cells.
FIG. 27 shows micrographs showing the time- and concentration-dependent cytotoxicity of the conjugate Orn9-K-FAM of the present disclosure against normal human umbilical vein endothelial cells.
As shown in Figures 26 and 27, the complex containing the microbial membrane permeabilizing agent of the present disclosure was found to have low and limited cytotoxicity against normal human umbilical vein endothelial cells in a time- and concentration-dependent manner.

Claims (17)

A bacterial membrane permeabilizing agent comprising at least one of a first membrane-permeable peptide, in which n=2 and the first peptide is composed of 5 to 19 consecutive amino acid residues of formula (I), or in which n=1 and the second peptide is composed of amino acid residues of formula (II).

[In formula (II), n is independently an integer of 1 to 3. In formulas (I) and (II), the amino acid residue may be either the (R) or (S) configuration. In formula (II), m is an integer of 2 to 4, and ^R1 and ^R2 each independently represent a hydrophobic substituent derived from the side chain of an amino acid having a hydrophobic index of -2 or more. In formulas (I) and (II), the moiety represented by * represents a binding site to an adjacent moiety.] The bacterial membrane permeabilizing agent according to claim 1, wherein the first membrane-permeabilizing peptide consists of 5 to 19 consecutive 2,4-diaminobutanoic acid (Dab) residues. The bacterial membrane permeabilizing agent according to claim 1 or 2, wherein the first membrane-permeable peptide consists of 5, 6, 7, 8, 9, 10, 11 or 12 consecutive (S) -2,4-diaminobutanoic acid (Dab) residues. The bacterial membrane permeabilizing agent according to any one of claims 1 to 3, wherein 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 formula (I). The bacterial membrane permeabilizing agent according to any one of claims 1 to 4, wherein the first membrane-permeable peptide has two blocks each consisting of 4 to 5 independently selected amino acid residues of formula (I), and contains one or two consecutive amino acid residues other than the amino acid residues of formula (I) at at least one position between the two blocks. The bacterial membrane permeabilizing agent according to claim 5, wherein the amino acid residue other than the amino acid residue of formula (I) includes a lysine (Lys) residue having a labeling molecule as a substituent, or an arginine (Arg) residue having a labeling molecule as a substituent. The bacterial membrane permeabilizing agent according to any one of claims 1 to 6, wherein the bacterium is a bacterium of the family Enterobacteraceae, the genus Synechocystis, the genus Pseudomonas, the genus Acinetobacter, or the genus Bacillus. A complex comprising a bacterial membrane permeabilizing agent according to any one of claims 1 to 7 and a compound. The complex according to claim 8, wherein the compound comprises at least one selected from the group consisting of a protein, a peptide, a nucleic acid, a peptide nucleic acid, an inorganic substance, an organometallic compound, an organic compound, a polymer, a nanoparticle, a molecular assembly including a micelle and a liposome, a sugar, and a labeling substance. The complex according to claim 8 or claim 9, wherein the bacterial membrane permeabilizing agent and the compound are linked directly or via a linker. The complex according to claim 10, wherein the linker consists of 1 to 10 consecutive amino acid residues. A method for delivering a compound to a bacterial cell, comprising contacting the bacterial cell with a complex comprising the bacterial membrane permeabilizing agent according to any one of claims 1 to 7 and a compound. The method according to claim 12, wherein the compound comprises at least one selected from the group consisting of proteins, peptides, nucleic acids, peptide nucleic acids, inorganic substances, organometallic compounds, organic compounds, polymers, nanoparticles, sugars, and labeling substances, as well as molecular assemblies including micelles and liposomes. The method according to claim 12 or 13, wherein the bacterium is a bacterium of the family Enterobacteraceae, the genus Synechocystis, the genus Pseudomonas, the genus Acinetobacter, or the genus Bacillus. A bactericide comprising the bacterial membrane permeabilizing agent according to any one of claims 1 to 7. An antibacterial agent comprising the bacterial membrane permeabilizing agent according to any one of claims 1 to 7. A labeling agent comprising the bacterial membrane permeabilizing agent according to any one of claims 1 to 7.