Epsilon-poly-L-lysine derivative with click functional group, and preparation method and application thereof

Abstract

The present invention provides: a compound having a structure represented by general formula (1) (in general formula (1), n is 2 to 100; p represents a (C1-C5) alkylene group substituted by an amino group; q is represented by general formula (2) (in general formula (2), X represents an oxygen atom, Y1 and Y2 represent alkylene groups having 1 to 6 carbon atoms, and m is an integer of 1 to 30; z represents a click functional group. # imgabs0 #

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201880055002.7 filed on February 24, 2020 and invention name “ε-poly-L-lysine derivatives with click functional groups, preparation methods thereof and uses thereof”. Technical Field

[0002] The present invention relates to an ε-poly-L-lysine derivative whose carboxyl terminal is modified with a click functional group, a method for producing the same, and its use or utilization for simultaneously improving biomembrane permeability and water solubility of a pharmacological substance, polycation modification of an industrial substrate, and antimicrobial coating. Background Art

[0003] ε-Poly-L-lysine (hereinafter referred to as ε-PL) is a linear amino acid homopolymer of 10 to 35 residues of L-lysine, which is a proteinogenic amino acid, and is characterized by a structure in which the carboxyl group of L-lysine and the amino group at the ε position are connected by an isopeptide bond. ε-PL is produced by a variety of microorganisms including actinomycetes, and its peptide chain length (the number of L-lysine) varies depending on the producing bacteria (Non-patent Documents 1 to 4). In the first report on ε-PL, ε-PL of 25 to 35 residues was identified from the culture fluid of the actinomycete Streptomyces albulus No. 346-D strain, and later, ε-PL with a shorter chain length than this ε-PL (short-chain ε-PL) was identified. However, ε-PL composed of more than 36 L-lysine residues has not yet been found in nature. The α-amino group (-NH2) of ε-PL is protonated (-NH3 + ), so ε-PL exists as a super water-soluble polycationic peptide, and shows antibacterial activity against a wide range of microbial species, just like synthetic polycationic antibacterial agents (Non-patent Document 4). ε-PL produced by Streptomyces albulus No. 346-D strain and composed of 25 to 35 residues is used as a food preservative or food preservative because of its wide range of antibacterial activity and high safety. In addition, it is also expected to be a biodegradable natural antibacterial agent with high safety and low environmental load in not only the food industry, but also in the chemical industry, medical field, etc.

[0004] The inventors of the present invention have paid attention to the fact that polycationic compounds show antibacterial activity, have high polarity and high biomembrane permeability, and have considered whether it is possible to expect to improve the water solubility and biomembrane permeability of pharmacological substances at the same time by combining ε-PL (hereinafter referred to as ε-PL modification) on pharmacological substances (non-patent document 4). In addition, it is considered that if the surfaces of various medical substrates, industrial substrates, etc. (hereinafter collectively referred to as industrial substrates) such as plastics, metals, wood, paper, and fibers can be modified with ε-PL, whether it is possible to impart various ideal functions such as antibacterial activity, cultured cell adhesion effect, anti-drip effect, and antistatic function due to polycationic properties. However, so far, there is no report on the ε-PL modification of pharmacological substances. However, regarding the ε-PL modification of industrial substrates, a method of utilizing the electrostatic interaction of the polycationic property (positive charge) of ε-PL and the surface negative charge of the industrial substrate has been reported (patent document 1). However, the stability and durability of the chemical modification method by non-covalent bonds are extremely low, and no practical ε-PL modification method has been established. In addition, it can only be applied to materials with negative surface charge, and it cannot be called a universal modification method. In addition, the polycationic property (positive charge) of ε-PL is an important chemical property for exerting antibacterial activity, cultured cell adhesion effect, anti-drip effect and antistatic function. The ε-PL modification of industrial substrates using electrostatic interaction leads to a decrease in the polycationic property of ε-PL, so there is a problem that the expected function cannot be fully exerted. The inventors have developed an ε-PL modification method that utilizes covalent bonds instead of electrostatic interactions, and at the same time found a strong effect. As a method for producing ε-PL, a method for producing it using Streptomyces albulus No.346-D strain (NBRC14147) isolated from nature is known (Patent Document 2). In addition, as a strain other than Streptomyces albulus, a method for producing it using Streptomyces noursei (Patent Document 3) and Streptomyces sp.SP-72 (Patent Document 4) is known. In addition, a method for producing short-chain ε-PL using Streptomyces sp. SP-66 (Patent Document 5) is known. In order to completely preserve the polycationic properties of ε-PL, chemical modification of pharmacological substances and industrial substrates through more stable covalent bonds requires organic synthetic chemistry using the only remaining carboxyl group in ε-PL. However, a high level of organic synthetic chemistry technology is required to protect all α-amino groups (-NH2) of ε-PL with protective groups, and no successful cases have been reported.Click Chemistry is an organic synthesis technology that utilizes a functional group with high reaction specificity (hereinafter referred to as a click functional group) without using a protecting group. It is a chemical technology that is used not only in the chemical industry but also in other fields. Although it is considered that if a click functional group can be introduced into the carboxyl group of ε-PL, it is extremely easy to modify ε-PL with pharmacological substances and industrial substrates, but even in the case of introducing a click functional group into the carboxyl group of ε-PL, a high-level organic synthesis chemistry technology is required to protect all α-amino groups (-NH2) of ε-PL with a protecting group, and no successful case has been reported. In addition, the production of ε-PL derivatives into which click functional groups are introduced by ε-PL producing microorganisms is also unknown.

[0005] Prior art literature

[0006] Patent Document Patent Document 1: Japanese Patent No. 3111216

[0007] Patent Document 2: Japanese Patent Publication No. 59-20359

[0008] Patent Document 3: Japanese Patent Application Laid-Open No. 1-187090

[0009] Patent Document 4: Japanese Patent Application Publication No. 2000-69988

[0010] Patent Document 5: Japanese Patent Application Publication No. 2001-017159

[0011] Non-patent literature Non-patent literature 1: Shima S, Sakai H. 1977. Polylysine produced by Streptomyces. 1977.

[0012] Non-patent literature 2: Oppermann-Sanio FB, Steinbuechel A. 2002. Occurrence, functions and biosynthesis of polyamides in microorganisms and biotechnological production. 2002.

[0013] Non-patent literature 3: Takehara M, Hirohara H. 2010. Amino-acid homopolymersoccurring in nature, p. 1-22. In Hamano Y (ed.), Microbiology Monographs, vol. 15. Springer, New York.

[0014] Non-patent document 4: Hamano Y. 2010. Amino-acid homopolymers occurring innature: Biochemistry and enzymology of poly-epsilon-L-lysine, p23-44. In Hamano (ed), Microbiology Monographs, vol. 15. Springer, New York. Summary of the invention

[0015] Problem to be solved by the invention The object of the present invention is to provide a compound having a structure represented by the general formula (1) or a chemically or pharmaceutically acceptable salt thereof and a method for producing these compounds or their chemically or pharmaceutically acceptable salts (hereinafter chemically acceptable salts and pharmaceutically acceptable salts are collectively referred to as salts).

[0016]

[0017] (wherein n is 2 to 100;

[0018] P represents a (C1-C5) alkylene group substituted with an amino group;

[0019] Q is a structure represented by the following general formula (2) or the following general formula (3),

[0020]

[0021] (In the above general formula (2), X represents an oxygen atom, an imino group, a sulfur atom, NHCO or CONH, and Y 1 and Y 2 may be the same or different, and represent an alkylene group having 1 to 6 carbon atoms, and m is an integer from 1 to 30.

[0022]

[0023] (In the above general formula (3), X represents an oxygen atom, an imino group, a sulfur atom, NHCO or CONH, Y represents an alkylene group having 1 to 6 carbon atoms, and m is an integer from 1 to 30.)

[0024] Z represents a click functional group. )

[0025] Another object of the present invention is to provide an ε-poly-L-lysine (ε-PL) derivative having a structure represented by the general formula (4) or a chemically or pharmaceutically acceptable salt thereof, and a method for producing these compounds or their chemically or pharmaceutically acceptable salts (hereinafter chemically acceptable salts and pharmaceutically acceptable salts are collectively referred to as salts).

[0026]

[0027] (wherein n is an integer from 2 to 100;

[0028] m is an integer from 1 to 30;

[0029] X is an oxygen atom or an imino group;

[0030] Y represents an alkylene group having 1 to 6 carbon atoms;

[0031] Z represents a click functional group. )

[0032] Another object of the present invention is to provide a pharmacological substance that has improved biomembrane permeability and water solubility by introducing the above-mentioned compound (including ε-PL derivatives) not only into low-molecular compounds and medium-molecular compounds such as antibacterial or anticancer substances, but also into pharmacological substances such as high-molecular compounds such as proteins (including enzymes) through covalent bonds, and a method for producing the same. In addition, another object of the present invention is to provide the use of the above-mentioned compound (including ε-PL derivatives) for improving the biomembrane permeability of pharmacological substances.

[0033] In addition, by using the above-mentioned compounds (including ε-PL derivatives) on the surface of industrial substrates through covalent bonds, the above-mentioned compounds are introduced into various industrial substrates, thereby imparting various functions such as antibacterial activity brought by polycationic properties, cultured cell adhesion effect, anti-drip effect and antistatic function to the industrial substrates.

[0034] Means used to solve problems

[0035] The present invention includes the following inventions in order to solve the above-mentioned problems.

[0036] [1] a compound having a structure represented by the general formula (1) or a salt thereof,

[0037]

[0038] Wherein, n is 2 to 100;

[0039] P represents a (C1-C5) alkylene group substituted with an amino group;

[0040] Q is a structure represented by the following general formula (2) or the following general formula (3),

[0041]

[0042] In the above general formula (2), X represents an oxygen atom, an imino group, a sulfur atom, NHCO or CONH, and Y 1 and Y 2may be the same or different, and represent an alkylene group having 1 to 6 carbon atoms, an oxygen atom or an imino group, and m is an integer from 1 to 30;

[0043]

[0044] In the above general formula (3), X represents an oxygen atom, an imino group, a sulfur atom, NHCO or CONH, Y represents an alkylene group having 1 to 6 carbon atoms, an oxygen atom or an imino group, and m is an integer from 1 to 30;

[0045] Z represents a click functional group.

[0046] [2] The compound or salt thereof according to [1] above, which is represented by the general formula (4):

[0047]

[0048] Wherein, n is an integer from 2 to 100;

[0049] m is an integer from 1 to 30;

[0050] X is an oxygen atom or an imino group;

[0051] Y represents an alkylene group having 1 to 6 carbon atoms;

[0052] Z represents a click functional group.

[0053] [3] The compound or salt thereof according to [1] or [2] above, wherein the click functional group is an azido group, an alkenyl group, an alkynyl group, a nitrile group, a thiol group, a maleimide group, an epoxy group, an aziridine group, a thioethane group or a triazole group.

[0054] [4] A method for producing the compound or a salt thereof according to [1] above, comprising:

[0055] A step of culturing a bacterium producing ε-poly-L-lysine, ε-poly-D-lysine, ε-poly-L-β-lysine, ε-poly-D-β-lysine, δ-poly-L-ornithine, δ-poly-D-ornithine, δ-poly-L-β-ornithine, δ-poly-D-β-ornithine, γ-poly-L-diaminobutyric acid, γ-poly-D-diaminobutyric acid, β-poly-L-diaminopropionic acid or β-poly-D-diaminopropionic acid in the presence of a compound represented by the general formula (2a) or the general formula (3a) or a salt thereof; and

[0056] a step of collecting the produced compound or salt thereof described in [1],

[0057] The general formula (2a) is

[0058]

[0059] In the above general formula (2a), X, Y 1 , Y 2 , Z and m have the same meanings as in the above general formula (1); general formula (3a) is

[0060]

[0061] In the above general formula (3a), X, Y, Z and m have the same meanings as in the above general formula (1).

[0062] [5] A method for producing the compound or a salt thereof according to [2], comprising:

[0063] a step of culturing an ε-PL-producing bacterium in the presence of a compound represented by the general formula (3a) or a salt thereof, and

[0064] a step of collecting the produced compound or salt thereof described in [2],

[0065] The general formula (3a) is

[0066]

[0067] In the above general formula (3a), X represents an oxygen atom or an imino group, Y represents an alkylene group having 1 to 6 carbon atoms, Z represents a click functional group, and m represents an integer of 1 to 30.

[0068] [6] A pharmacologically active substance, wherein the compound described in [1] or a pharmaceutically acceptable salt thereof is introduced via a chemical bond.

[0069] [7] A pharmacologically active substance, wherein a compound represented by the general formula (4) or a pharmaceutically acceptable salt thereof is introduced via a chemical bond,

[0070]

[0071] Wherein, n is an integer from 2 to 100,

[0072] m is an integer from 1 to 30,

[0073] X represents an oxygen atom or an imino group,

[0074] Y represents an alkylene group having 1 to 6 carbon atoms,

[0075] Z represents a click functional group.

[0076] [8] The substance according to [6] or [7] above, wherein the pharmacologically active substance is at least one of an antibacterial agent, an antimicrobial and antibiotic agent, and an anticancer agent.

[0077] [9] The substance according to [8] above, wherein

[0078] Antimicrobial agents are (A) antifungal agents;

[0079] The antimicrobial antibiotic is (B) a β-lactam antibiotic, (C) an aminoglycoside antibiotic, (D) a tetracycline antibiotic, or (E) a peptide antibiotic, or (F) an antimicrobial antibiotic capable of introducing a click functional group organically or biochemically; or

[0080] The anticancer agent is (G) an anticancer agent into which a click functional group can be introduced organically or biochemically.

[0081]

[10] The substance described in [9] above, wherein

[0082] (A) the antifungal agent is (a-1) a polyene antibiotic selected from amphotericin B (hereinafter referred to as AmpB), nystatin, chloramphenicol and pimacin, (a-2) micafungin, (a-3) itraconazole, (a-4) ketoconazole, (a-5) fluconazole, (a-6) miconazole, or (a-7) flucytosine;

[0083] (B) the β-lactam antibiotic is (b-1) penicillin, (b-2) ampicillin, (b-3) talancillin, (b-4) bacillin, (b-5) cephalosporin C, (b-6) cephalexin, (b-7) cephradine, (b-8) cephamycin, or (b-9) imipenem;

[0084] (C) the aminoglycoside antibiotic is (c-1) gentamicin, (c-2) kanamycin, (c-3) tobramycin, (c-4) amikacin, (c-5) arbekacin, or (c-6) nucleomycin;

[0085] (D) the tetracycline antibiotic is (d-1) oxytetracycline, or (d-2) tetracycline,

[0086] (E) the peptide antibiotic is (e-1) bacitracin, (e-2) gramicidin, (e-3) polymyxin B, (e-4) vancomycin, or (e-5) teicoplanin;

[0087] (F) an antimicrobial antibiotic having an amino group, an imino group, a carboxyl group, a hydroxyl group, a carbonyl group, a thiol group, a phosphate group, an aldehyde group, etc. as an antimicrobial antibiotic into which a click functional group can be introduced organically or biochemically; or

[0088] (G) As an anticancer agent capable of introducing a click functional group organically or biochemically, it is an anticancer agent having any one of an amino group, an imino group, a carboxyl group, a hydroxyl group, a carbonyl group, a thiol group, a phosphate group, an aldehyde group, etc., for example, (f-1) actinomycin D, (f-2) mitomycin, (f-3) an anthracycline antitumor drug selected from doxorubicin (hereinafter referred to as Dox), daunorubicin, and aclarubicin, (f-4) bleomycin, or (f-5) cisplatin.

[0089]

[11] The substance according to [6] or [7] above, wherein the pharmacological agent is at least one of a protein such as an enzyme or an antibody and a gene.

[0090]

[12] The substance according to

[11] above, wherein the click functional group can be introduced into the protein or gene organically or biochemically.

[0091]

[13] The method for producing a pharmacologically active substance according to the above [7], characterized in that the compound represented by the general formula (4) or a pharmaceutically acceptable salt thereof described in the above [7] is introduced into the pharmacologically active substance via a chemical bond.

[0092]

[14] The substance according to any one of [7] to

[10] above, which is AmpB modified with ε-PL by click chemistry (hereinafter referred to as ε-PL-DBCO-AmpB) represented by the general formula (5),

[0093]

[0094] Wherein n is an integer from 2 to 100.

[0095]

[15] The substance according to any one of [7] to

[10] above, which is Dox (hereinafter referred to as ε-PL-DBCO-Dox) modified by click chemistry and represented by the general formula (5-2),

[0096]

[0097] Wherein n is an integer from 2 to 100.

[0098]

[16] The substance according to any one of [7] to

[10] above, which is mAG (hereinafter referred to as ε-PL-DBCO-mAG) modified with ε-PL by click chemistry and represented by the general formula (5-3),

[0099]

[0100] Wherein, n is an integer from 2 to 100, formula (5-3)'

[0101]

[0102] The group shown is formula (5-3)"

[0103]

[0104] The monomeric Azami-Green (hereinafter referred to as mAG) fluorescent protein shown is a group formed by removing a hydrogen atom.

[0105]

[17] Use of a compound represented by the general formula (2a) or (3a) or a salt thereof for producing a compound represented by the general formula (1) or a salt thereof as described in [1] above.

[0106]

[18] Use of a compound represented by the general formula (3a) or a salt thereof for producing a compound represented by the general formula (4) or a salt thereof as described in [7] above,

[0107] General formula (3a):

[0108]

[0109] X represents an oxygen atom or an imino group, Y represents an alkylene group having 1 to 6 carbon atoms, Z represents a click functional group, and m represents an integer of 1 to 30.

[0110]

[19] A method for improving the biomembrane permeability of a pharmacological substance, characterized in that a compound represented by the general formula (1) or a salt thereof is introduced into the pharmacological substance; or a use of a compound represented by the general formula (1) or a salt thereof for the manufacture of a pharmacological substance that improves the biomembrane permeability.

[0111]

[20] A method for improving biomembrane permeability of a pharmacological substance, characterized in that a compound represented by the general formula (4) described in [7] above or a salt thereof is introduced into the pharmacological substance; or a compound represented by the general formula (4) or a salt thereof is used to prepare a pharmacological substance that improves biomembrane permeability.

[0112]

[21] A method for improving the water solubility of a pharmacological substance, characterized by introducing a compound represented by the general formula (1) or a salt thereof into the pharmacological substance.

[0113]

[22] Use of a compound represented by the general formula (1) or a salt thereof for producing a pharmacological substance having improved water solubility.

[0114]

[23] A method for improving the water solubility of a pharmacological substance, characterized in that the compound represented by the general formula (4) or a salt thereof as described in [7] above is introduced into the pharmacological substance; or a use of a compound represented by the general formula (4) for producing a pharmacological substance with improved water solubility.

[0115] Effect of the Invention The hyperactivity of drug excretion function in pathogenic bacteria or cancer cells is associated with drug resistance to therapeutic drugs. If the biomembrane permeability of pharmacological substances such as drugs can be improved, not only can the drug resistance mechanism be avoided, but also the pharmacological substances that cannot be used due to drug resistance can be reused, and the physiological activity can be enhanced. The general countermeasure for improving membrane permeability is to hydrophobize the pharmacological substances. However, on the other hand, the reduction in water solubility will produce new problems such as unexpected side effects and complicated preparation of preparations. Therefore, the inventors consider that if a functional low-molecular chemical modification technology that improves biomembrane permeability and water solubility can be developed at the same time, the speed of drug development required by modern society can be further accelerated.

[0116] High molecular weight compounds such as proteins (including enzymes) usually do not penetrate the cell membrane. This property is important in maintaining the dynamic balance of cells. On the other hand, it can be said that it is difficult to directly introduce these molecules from outside the cell into the interior for functional analysis, control or medical purposes. Currently, methods such as microinjection, electroporation, etc. that instantly destroy the cell membrane and introduce, methods using liposomes to introduce, etc. are used. However, due to the high efficiency of introduction and the damage to cells, satisfactory results may not be obtained. On the other hand, attention has been paid to the excellent cell membrane permeability of basic (polycationic) peptides, and attempts have been made to modify proteins (including enzymes) with polycationic peptides and directly introduce them into cells (non-patent literature, SR, Schwarze et al., Science, 285, 1569, 1999). In fact, many examples of preparing polycation-modified proteins as chemical crosslinkers or fusion proteins with polycationic peptides, which can control cell functions only by adding to cell culture fluid, are recognized as one of the cell biochemical methods, and are also receiving attention as a new delivery method for biopharmaceuticals (antibody medicines, etc.). The inventors of the present invention have conceived of polycations including ε-PL as compounds that can solve these problems. ε-PL is a polycationic natural compound that has high polarity, super water solubility, and high biomembrane permeability. Acidic phospholipids are exposed on the surface of bacterial cell membranes, and there are a large number of sugar chains containing many sulfate groups or carboxyl groups on the surface of animal cells, so their surface is negatively charged. The inventors of the present invention have conceived of polycations containing positively charged ε-PL, which are attracted to the negatively charged surface and easily pass through the cell membrane and move into the cell. That is, in the present disclosure, polycations refer to, for example, compounds with polycationic properties. For example, polycations are formed on the amino group of the lysine group of polylysine, and in vivo, for example, H is added. + And generated.

[0117] In addition, similar effects can be obtained with natural polycation analogs other than ε-PL, such as γ-poly-L-diaminobutyric acid, γ-poly-D-diaminobutyric acid, and β-poly-L-diaminopropionic acid.

[0118] The present inventors have found that the compounds described in [1] above, including ε-PL derivatives represented by the general formula (1), can be easily bound to various pharmacological substances via stable covalent bonds by click chemistry. Generally, compounds with high water solubility have low biomembrane permeability. Although the polycations including ε-PL have very high water solubility, the biomembrane permeability is also very high due to their polycationic properties. Thus, one of the important features of the present invention is a simple and effective new method for producing compounds required for a simple method for modifying polycations including ε-PL, wherein the method for modifying polycations including ε-PL improves both the biomembrane permeability and water solubility of pharmacological substances.

[0119] In addition, in polymer compounds such as proteins (including enzymes), it is possible to impart biomembrane permeability by polycationization through click chemistry. Through the present disclosure, a new manufacturing method for directly delivering polymer compounds into cells can be provided. In addition, click chemistry has been fully established in the past, and the present invention can be operated in the same way.

[0120] In the present invention, by treating with a pharmacologically active substance introduced with the compound represented by the above-mentioned general formula (1) (including the compound represented by the general formula (4) in this specification) or its salt, various industrial substrates (for example, medical equipment materials made of plastic products, fibers, etc., functional materials, etc.) can be polycationized (including ε-PL modification), and the industrial equipment materials can be endowed with excellent antibacterial activity, cultured cell adhesion effect, water immersion treatment, anti-drip effect, and antistatic function. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1A The results of chromatographic analysis of the culture supernatant of Streptomyces albulus NBRC14147 by ESI-TOF-MS / HPLC analysis are shown. The upper figure shows the analysis results of the culture medium without additives, the middle figure shows the analysis results of the culture medium with the addition of ε-PL-PEG-azide, and the lower figure shows the analysis results of the culture medium with the addition of ε-PL-PEG-alkyne.

[0122] Figure 1B The results of ESI-TOF-MS of ε-PL (upper panel), ε-PL-PEG-azide (middle panel), and ε-PL-PEG-alkyne (lower panel) are shown.

[0123] Figure 2A The composition of ε-PL-PEG-azide composed of 17-26mer ε-PL produced by ε-PL producing bacteria Streptomyces albulus NBRC14147 is shown.1 Results of structure determination by H-NMR.

[0124] Figure 2B Figure 2 shows the production of ε-PL-PEG-alkyne by the ε-PL producing bacterium Streptomyces albulus NBRC14147. 1 Results of H-NMR structure determination.

[0125] Figure 3A The graph shows the results of the ESI-TOF-MS / HPLC analysis of a chromatogram of a reaction solution in which ε-PL-DBCO-FAM was synthesized by click chemistry.

[0126] Figure 3B The results of ESI-TOF-MS of synthesized ε-PL-DBCO-FAM are shown.

[0127] Figure 4 The results of a biomembrane permeability test using HeLa cells using DBCO-PEG4-5 / 6-FAM represented by formula (12) and ε-PL-DBCO-FAM represented by general formula (13) are shown.

[0128] Figure 5 The results of confocal laser microscopy observation of biomembrane permeability using HeLa cells for ε-PL-DBCO-FAM represented by general formula (13) are shown. Biomembranes and nuclei were stained with Alexa594WGA (wheat germ agglutinin) and 4',6-diamidino-2-phenylindole (DAPI), respectively.

[0129] Fig. 6A The graph shows the results of the ESI-TOF-MS / HPLC analysis of a chromatogram of a reaction solution in which ε-PL-DBCO-AmpB was synthesized by click chemistry.

[0130] Figure 6B The results of ESI-TOF-MS of synthesized ε-PL-DBCO-AmpB are shown.

[0131] Figure 7 The water solubility of ε-PL-DBCO-AmpB is shown.

[0132] Fig. 8A The graph shows the results of the ESI-TOF-MS / HPLC analysis of a chromatogram of a reaction solution in which ε-PL-DBCO-Doc was synthesized by click chemistry.

[0133] Figure 8B The results of ESI-TOF-MS of synthesized ε-PL-DBCO-Doc are shown.

[0134] Fig.9A The graphs show the chromatogram of ESI-TOF-MS / HPLC analysis of mAG represented by general formula (5-3) (left), the mass spectrum of ESI-TOF-MS (middle), and the results of deconvolution of the obtained mass spectrum (right).

[0135] Fig. 9B Shown are a chromatogram of ESI-TOF-MS / HPLC analysis of DBCO-mAG (left), a mass spectrum of ESI-TOF-MS (middle), and the results of deconvolution of the obtained mass spectrum (right).

[0136] Fig. 9C Shown are the chromatogram (left) of ESI-TOF-MS / HPLC analysis of ε-PL-DBCO-mAG represented by the general formula (5-3), the mass spectrum of ESI-TOF-MS (middle), and the results of deconvolution of the obtained mass spectrum (right).

[0137] Fig.10 The results of a biomembrane permeability test using HeLa cells for mAg represented by the general formula (5-3)" and ε-PL-DBCO-mAG represented by the general formula (5-3) are shown.

[0138] Fig.11 The results of the confocal laser microscopy observation of the biomembrane permeability evaluation of ε-PL-DBCO-mAG represented by the general formula (5-3) using HeLa cells are shown. The biomembrane was stained with Alexa594WGA (wheat germ agglutinin).

[0139] Fig. 12A The results of verifying the chemical stability of the ester bond of the ε-PL-alkyne represented by the general formula (15) are shown. The ε-PL-alkyne was dissolved in a Tris-HCl buffer (pH 8.0) at a concentration of 1 mM, heated at 60°C for 5 minutes, and then analyzed by ESI-TOF-MS / HPLC. # represents the signal of the ε-PL-alkyne, and * represents the signal of the ε-PL generated by the hydrolysis of the ester bond of the ε-PL-alkyne.

[0140] Fig. 12B The results of verifying the chemical stability of the ester bond of ε-PL-PEG-alkyne represented by general formula (11) are shown. ε-PL-PEG-alkyne was dissolved in Tris-HCl buffer (pH 8.0) at a concentration of 1 mM, heated at 60°C for 5 minutes, and analyzed by ESI-TOF-MS / HPLC. + represents the signal of ε-PL-PEG-alkyne, and * represents the signal of ε-PL generated by hydrolysis of the ester bond of ε-PL-PEG-alkyne. DETAILED DESCRIPTION

[0141] In the present invention, P in the compound represented by the general formula (1) is a (C1-C5) alkylene group substituted by 1 or 2 or more amino groups. The (C1-C5) alkylene group may be linear or branched, and is preferably linear. P in the compound represented by the general formula (1) is preferably a (C1-C5) alkylene group substituted by 1 amino group, and specifically, CH2CH2CH2CH2C * H(NH2),CH2CH2CH2C * H(NH2)CH2、CH2CH2C * H(NH2)CH2CH2、CH2C * H(NH2)CH2CH2CH2、C * H(NH2)CH2CH2CH2CH2, CH2CH2CH2C * H(NH2),CH2CH2C * H(NH2)CH2、CH2C * H(NH2)CH2CH2、C * H(NH2)CH2CH2CH2、CH2CH2C * H(NH2), CH2C * H(NH2)CH2、C * H(NH2)CH2CH2、CH2C * H(NH2) or C * H(NH2)CH2, etc. Here, * represents an asymmetric carbon, and may be of R or S type. In the present invention, with respect to the compound represented by the general formula (1), the imino group represented by X is represented by "NR", and as R, H or a suitable substituent may be mentioned, for example, a lower alkyl group having 1 to 6 carbon atoms such as methyl and ethyl, an aryl group such as phenyl, and for example, a heterocyclic group having 1 to 3 heteroatoms such as furyl, pyridyl, and thienyl. Among them, R is preferably a 5- to 6-membered heterocyclic group.

[0142] As Y, Y 1 or Y 2The alkylene group having 1 to 6 carbon atoms shown in the figure includes, for example, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, hexylene, etc. Among them, Y is preferably an alkylene group having 2 to 6 carbon atoms. The alkylene group may be substituted by a common substituent, and examples of such substituents include halogen atoms (e.g., bromine atoms, chlorine atoms, fluorine atoms, etc.), hydroxyl groups, amino groups, mercapto groups, carboxyl groups, sulfo groups, etc. The alkylene group may contain, for example, an insertion group such as -O-, -S-, -S-S-, or a silyl group. As the click functional group in the present invention, for example, an azido group (-N3), an alkenyl group (e.g., -CH=CH2, -CH=CH-CH3), an alkynyl group (e.g., -C≡CH, -C≡C-CH3), a nitrile group, a thiol group, a maleimide group, an epoxy group, an aziridine group, a thioethane group, a triazolyl group, etc.

[0143] In each of the general formulae (1), (4), (5), (5-2) and (5-3) and the general formulae (6), (7), (10), (11), (13) and (15) described later, n may be any one of 2 to 100, preferably about 10 to 40, and more preferably about 9 to 30. n varies depending on the type of bacteria to be cultured and the culture conditions of the bacteria (e.g., pH, temperature, time, etc.), and therefore cannot be generalized, but generally, compounds wherein n is 2 to 35, compounds wherein n is 25 to 35, compounds wherein n is 9 to 18, or compounds wherein n is 17 to 26 are preferred.

[0144] In the general formula (1) or (4), m may be any one of 1 to 30, and is preferably about 2 to 20. When m is 2 or more, the biomembrane permeability improvement effect or water solubility improvement effect of the pharmacological action substance introduced into the compound represented by the general formula (1) or a salt thereof can be further improved, and the compound can have high chemical stability.

[0145] Specific examples of the compound represented by the general formula (1) (where Q represents a structure represented by the general formula (2)) are shown below. [Table 1]

[0146] Compound No. X <![CDATA[Y 1 ]]> <![CDATA[Y 2 ]]> Z m n 1 O <![CDATA[CH2]]> O <![CDATA[N3]]> 4 30 2 O <![CDATA[CH2CH2]]> NH <![CDATA[N3]]> 12 10 3 O <![CDATA[CH2CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[N3]]> 4 20 4 O <![CDATA[CH2CH2CH2CH2]]> O <![CDATA[N3]]> 4 40 5 O <![CDATA[CH2CH2CH2CH2CH2]]> NH <![CDATA[N3]]> 4 50 6 O <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[N3]]> 2 60 7 NH <![CDATA[CH2]]> O <![CDATA[N3]]> 30 70 8 NH <![CDATA[CH2CH2]]> NH <![CDATA[N3]]> 10 80 9 NH <![CDATA[CH2CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[N3]]> 4 90 10 NH <![CDATA[CH2CH2CH2CH2]]> O <![CDATA[N3]]> 4 100 11 NH <![CDATA[CH2CH2CH2CH2CH2]]> NH <![CDATA[N3]]> 4 2 12 NH <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[N3]]> 4 25 13 <![CDATA[NCH3]]> <![CDATA[CH2]]> O <![CDATA[N3]]> 20 30 14 <![CDATA[NCH3]]> <![CDATA[CH2CH2]]> NH <![CDATA[N3]]> 8 35 15 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[N3]]> 4 25 16 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2]]> O <![CDATA[N3]]> 4 30 17 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2]]> NH <![CDATA[N3]]> 4 35 18 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[N3]]> 4 25 19 NHCO <![CDATA[CH2]]> O <![CDATA[N3]]> 16 30 20 NHCO <![CDATA[CH2CH2]]> NH <![CDATA[CH=CH2]]> 6 35 21 NHCO <![CDATA[CH2CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[CH=CHCH3]]> 4 25 22 NHCO <![CDATA[CH2CH2CH2CH2]]> O C≡CH 4 30 23 NHCO <![CDATA[CH2CH2CH2CH2CH2]]> NH <![CDATA[C≡CCH3]]> 4 35 24 NHCO <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[NCH3]]> Nitrile 4 25 25 CONH <![CDATA[CH2]]> O Thiol 14 30 26 CONH <![CDATA[CH2CH2]]> NH Maleimide 4 35 27 CONH <![CDATA[CH2CH2CH2]]> <![CDATA[NCH3]]> Epoxy 4 25 28 CONH <![CDATA[CH2CH2CH2CH2]]> O Aziridine 4 30 29 CONH <![CDATA[CH2CH2CH2CH2CH2]]> NH Ethyl sulfide 4 35 30 CONH <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[NCH3]]> Triazolyl 4 30

[0147] In the above Table 1, P represents CH2CH2CH2CH2C*H(NH2) (here the asymmetric carbon is of R type).

[0148] [Table 2]

[0149] Compound No. X <![CDATA[Y 1 ]]> <![CDATA[Y 2 ]]> Z m n 31 O <![CDATA[CH2]]> O <![CDATA[N3]]> 4 30 32 NH <![CDATA[CH2CH2]]> NH <![CDATA[N3]]> 10 80 33 NH <![CDATA[CH2CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[N3]]> 4 90 34 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2CH2]]> O <![CDATA[N3]]> 4 25 35 NHCO <![CDATA[CH2]]> NH <![CDATA[N3]]> 16 30 36 O <![CDATA[CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[CH=CH2]]> 6 35 37 O <![CDATA[CH2CH2CH2]]> O <![CDATA[CH=CHCH3]]> 4 25 38 O <![CDATA[CH2CH2CH2CH2CH2]]> NH C≡CH 4 30 39 O <![CDATA[CH2CH2CH2CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[C≡CCH3]]> 4 35 40 O <![CDATA[CH2]]> O Nitrile 4 25 41 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2]]> NH Thiol 14 30 42 <![CDATA[NCH3]]> <![CDATA[CH2CH2]]> <![CDATA[NCH3]]> Maleimide 4 35 43 CONH <![CDATA[CH2CH2CH2]]> O Epoxy 4 25 44 CONH <![CDATA[CH2CH2CH2CH2]]> NH Aziridine 4 30 45 CONH <![CDATA[CH2]]> <![CDATA[NCH3]]> Ethyl sulfide 4 35 46 CONH <![CDATA[CH2CH2CH2CH2CH2CH2]]> O Triazolyl 4 30 47 O <![CDATA[CH2]]> NH <![CDATA[N3]]> 4 30 48 NH <![CDATA[CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[N3]]> 10 80 49 NH <![CDATA[CH2CH2CH2]]> O <![CDATA[N3]]> 4 90 50 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2CH2]]> NH <![CDATA[N3]]> 4 25 51 NHCO <![CDATA[CH2]]> <![CDATA[NCH3]]> <![CDATA[N3]]> 16 30 52 O <![CDATA[CH2CH2]]> O <![CDATA[CH=CH2]]> 6 35 53 O <![CDATA[CH2CH2CH2]]> NH <![CDATA[CH=CHCH3]]> 4 25 54 O <![CDATA[CH2CH2CH2CH2CH2]]> <![CDATA[NCH3]]> C≡CH 4 30 55 O <![CDATA[CH2CH2CH2CH2CH2]]> O <![CDATA[C≡CCH3]]> 4 35 56 O <![CDATA[CH2]]> NH Nitrile 4 25 57 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2]]> <![CDATA[NCH3]]> Thiol 14 30 58 <![CDATA[NCH3]]> <![CDATA[CH2CH2]]> O Maleimide 4 35 59 CONH <![CDATA[CH2CH2CH2]]> NH Epoxy 4 25 60 CONH <![CDATA[CH2CH2CH2CH2]]> <![CDATA[NCH3]]> Aziridine 4 30

[0150] In the above Table 2, P in compound numbers 31 to 45 represents CH2CH2CH2C * H(NH2)CH2 (here, the asymmetric carbon is S-type), P in compound numbers 46 to 60 represents CH2CH2CH2C* H(NH2) (here, the asymmetric carbon is R type). [Table 3]

[0151] Compound No. X <![CDATA[Y 1 ]]> <![CDATA[Y 2 ]]> Z m n 61 O <![CDATA[CH2]]> O <![CDATA[N3]]> 4 30 62 NH <![CDATA[CH2CH2]]> NH <![CDATA[N3]]> 10 80 63 NH <![CDATA[CH2CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[N3]]> 4 90 64 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2CH2]]> O <![CDATA[N3]]> 4 25 65 NHCO <![CDATA[CH2]]> NH <![CDATA[N3]]> 16 30 66 O <![CDATA[CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[CH=CH2]]> 6 35 67 O <![CDATA[CH2CH2CH2]]> O <![CDATA[CH=CHCH3]]> 4 25 68 O <![CDATA[CH2CH2CH2CH2CH2]]> NH C≡CH 4 30 69 O <![CDATA[CH2CH2CH2CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[C≡CCH3]]> 4 35 70 O <![CDATA[CH2]]> O Nitrile 4 25 71 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2]]> NH Thiol 14 30 72 <![CDATA[NCH3]]> <![CDATA[CH2CH2]]> <![CDATA[NCH3]]> Maleimide 4 35 73 CONH <![CDATA[CH2CH2CH2]]> O Epoxy 4 25 74 CONH <![CDATA[CH2CH2CH2CH2]]> NH Aziridine 4 30 75 O <![CDATA[CH2]]> <![CDATA[NCH3]]> <![CDATA[N3]]> 4 30 76 NH <![CDATA[CH2CH2]]> O <![CDATA[N3]]> 10 80 77 NH <![CDATA[CH2CH2CH2]]> NH <![CDATA[N3]]> 4 90 78 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[N3]]> 4 25 79 NHCO <![CDATA[CH2]]> O <![CDATA[N3]]> 16 30 80 O <![CDATA[CH2CH2]]> NH <![CDATA[CH=CH2]]> 6 35 81 O <![CDATA[CH2CH2CH2]]> <![CDATA[NCH3]]> <![CDATA[CH=CHCH3]]> 4 25 82 O <![CDATA[CH2CH2CH2CH2CH2]]> O C≡CH 4 30 83 O <![CDATA[CH2CH2CH2CH2CH2]]> NH <![CDATA[C≡CCH3]]> 4 35 84 O <![CDATA[CH2]]> <![CDATA[NCH3]]> Nitrile 4 25 85 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2]]> O Thiol 14 30 86 <![CDATA[NCH3]]> <![CDATA[CH2CH2]]> NH Maleimide 4 35 87 CONH <![CDATA[CH2CH2CH2]]> <![CDATA[NCH3]]> Epoxy 4 25 88 CONH <![CDATA[CH2CH2CH2CH2]]> O Aziridine 4 30 89 CONH <![CDATA[CH2]]> NH Ethyl sulfide 4 35 90 CONH <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[NCH3]]> Triazolyl 4 30

[0152] In the above Table 3, P in compound numbers 61 to 70 represents CH2CH2C * H(NH2)CH2 (here, the asymmetric carbon is S-type), P in compound numbers 71 to 80 represents CH2CH2C * H(NH2) (here, the asymmetric carbon is R-type), and P in compound numbers 81 to 90 represents CH2C * H(NH2) (here, the asymmetric carbon is S-type). Specific examples of the compound represented by the general formula (1) (where Q represents a structure represented by the general formula (3)) are shown below.

[0153] [Table 4]

[0154] Compound No. X Y Z m n 91 O <![CDATA[CH2]]> <![CDATA[N3]]> 4 30 92 O <![CDATA[CH2CH2]]> <![CDATA[N3]]> 12 10 93 O <![CDATA[CH2CH2CH2]]> <![CDATA[N3]]> 4 20 94 O <![CDATA[CH2CH2CH2CH2]]> <![CDATA[N3]]> 4 40 95 O <![CDATA[CH2CH2CH2CH2CH2]]> <![CDATA[N3]]> 4 50 96 O <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[N3]]> 2 60 97 NH <![CDATA[CH2]]> <![CDATA[N3]]> 30 70 98 NH <![CDATA[CH2CH2]]> <![CDATA[N3]]> 10 80 99 NH <![CDATA[CH2CH2CH2]]> <![CDATA[N3]]> 4 90 100 NH <![CDATA[CH2CH2CH2CH2]]> <![CDATA[N3]]> 4 100 101 NH <![CDATA[CH2CH2CH2CH2CH2]]> <![CDATA[N3]]> 4 2 102 NH <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[N3]]> 4 25 103 <![CDATA[NCH3]]> <![CDATA[CH2]]> <![CDATA[N3]]> 20 30 104 <![CDATA[NCH3]]> <![CDATA[CH2CH2]]> <![CDATA[N3]]> 8 35 105 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2]]> <![CDATA[N3]]> 4 25 106 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2]]> <![CDATA[N3]]> 4 30 107 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2]]> <![CDATA[N3]]> 4 35 108 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[N3]]> 4 25 109 NHCO <![CDATA[CH2]]> <![CDATA[N3]]> 16 30 110 NHCO <![CDATA[CH2CH2]]> <![CDATA[CH=CH2]]> 6 35 111 NHCO <![CDATA[CH2CH2CH2]]> <![CDATA[CH=CHCH3]]> 4 25 112 NHCO <![CDATA[CH2CH2CH2CH2]]> C≡CH 4 30 113 NHCO <![CDATA[CH2CH2CH2CH2CH2]]> <![CDATA[C≡CCH3]]> 4 35 114 NHCO <![CDATA[CH2CH2CH2CH2CH2CH2]]> Nitrile 4 25 115 CONH <![CDATA[CH2]]> Thiol 14 30 116 CONH <![CDATA[CH2CH2]]> Maleimide 4 35 117 CONH <![CDATA[CH2CH2CH2]]> Epoxy 4 25 118 CONH <![CDATA[CH2CH2CH2CH2]]> Aziridine 4 30 119 CONH <![CDATA[CH2CH2CH2CH2CH2]]> Ethyl sulfide 4 35 120 CONH <![CDATA[CH2CH2CH2CH2CH2CH2]]> Triazolyl 4 30

[0155] In the above Table 4, P represents CH2CH2CH2CH2C * H(NH2) (here, the asymmetric carbon is of S type).

[0156] [Table 5]

[0157] Compound No. X Y Z m n 121 O <![CDATA[CH2]]> <![CDATA[N3]]> 4 30 122 NH <![CDATA[CH2CH2]]> <![CDATA[N3]]> 10 80 123 NH <![CDATA[CH2CH2CH2]]> <![CDATA[N3]]> 4 90 124 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[N3]]> 4 25 125 NHCO <![CDATA[CH2]]> <![CDATA[N3]]> 16 30 126 O <![CDATA[CH2CH2]]> <![CDATA[CH=CH2]]> 6 35 127 O <![CDATA[CH2CH2CH2]]> <![CDATA[CH=CHCH3]]> 4 25 128 O <![CDATA[CH2CH2CH2CH2CH2]]> C≡CH 4 30 129 O <![CDATA[CH2CH2CH2CH2CH2]]> <![CDATA[C≡CCH3]]> 4 35 130 O <![CDATA[CH2]]> Nitrile 4 25 131 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2]]> Thiol 14 30 132 <![CDATA[NCH3]]> <![CDATA[CH2CH2]]> Maleimide 4 35 133 CONH <![CDATA[CH2CH2CH2]]> Epoxy 4 25 134 CONH <![CDATA[CH2CH2CH2CH2]]> Aziridine 4 30 135 CONH <![CDATA[CH2]]> Ethyl sulfide 4 35 136 CONH <![CDATA[CH2CH2CH2CH2CH2CH2]]> Triazolyl 4 30 137 O <![CDATA[CH2]]> <![CDATA[N3]]> 4 30 138 NH <![CDATA[CH2CH2]]> <![CDATA[N3]]> 10 80 139 NH <![CDATA[CH2CH2CH2]]> <![CDATA[N3]]> 4 90 140 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[N3]]> 4 25 141 NHCO <![CDATA[CH2]]> <![CDATA[N3]]> 16 30 142 O <![CDATA[CH2CH2]]> <![CDATA[CH=CH2]]> 6 35 143 O <![CDATA[CH2CH2CH2]]> <![CDATA[CH=CHCH3]]> 4 25 144 O <![CDATA[CH2CH2CH2CH2CH2]]> C≡CH 4 30 145 O <![CDATA[CH2CH2CH2CH2CH2]]> <![CDATA[C≡CCH3]]> 4 35 146 O <![CDATA[CH2]]> Nitrile 4 25 147 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2]]> Thiol 14 30 148 <![CDATA[NCH3]]> <![CDATA[CH2CH2]]> Maleimide 4 35 149 CONH <![CDATA[CH2CH2CH2]]> Epoxy 4 25 150 CONH <![CDATA[CH2CH2CH2CH2]]> Aziridine 4 30

[0158] In the above Table 5, P in compound numbers 121 to 135 represents CH2CH2CH2C * H(NH2)CH2 (here, the asymmetric carbon is R type), P in compound numbers 136 to 150 represents CH2CH2CH2C * H(NH2) (here, the asymmetric carbon is S type). [Table 6]

[0159] Compound No. X Y Z m n 151 O <![CDATA[CH2]]> <![CDATA[N3]]> 4 30 152 NH <![CDATA[CH2CH2]]> <![CDATA[N3]]> 10 80 153 NH <![CDATA[CH2CH2CH2]]> <![CDATA[N3]]> 4 90 154 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[N3]]> 4 25 155 NHCO <![CDATA[CH2]]> <![CDATA[N3]]> 16 30 156 O <![CDATA[CH2CH2]]> <![CDATA[CH=CH2]]> 6 35 157 O <![CDATA[CH2CH2CH2]]> <![CDATA[CH=CHCH3]]> 4 25 158 O <![CDATA[CH2CH2CH2CH2CH2]]> C≡CH 4 30 159 O <![CDATA[CH2CH2CH2CH2CH2]]> <![CDATA[C≡CCH3]]> 4 35 160 O <![CDATA[CH2]]> Nitrile 4 25 161 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2]]> Thiol 14 30 162 <![CDATA[NCH3]]> <![CDATA[CH2CH2]]> Maleimide 4 35 163 CONH <![CDATA[CH2CH2CH2]]> Epoxy 4 25 164 CONH <![CDATA[CH2CH2CH2CH2]]> Aziridine 4 30 165 O <![CDATA[CH2]]> <![CDATA[N3]]> 4 30 166 NH <![CDATA[CH2CH2]]> <![CDATA[N3]]> 10 80 167 NH <![CDATA[CH2CH2CH2]]> <![CDATA[N3]]> 4 90 168 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2CH2]]> <![CDATA[N3]]> 4 25 169 NHCO <![CDATA[CH2]]> <![CDATA[N3]]> 16 30 170 O <![CDATA[CH2CH2]]> <![CDATA[CH=CH2]]> 6 35 171 O <![CDATA[CH2CH2CH2]]> <![CDATA[CH=CHCH3]]> 4 25 172 O <![CDATA[CH2CH2CH2CH2CH2]]> C≡CH 4 30 173 O <![CDATA[CH2CH2CH2CH2CH2]]> <![CDATA[C≡CCH3]]> 4 35 174 O <![CDATA[CH2]]> Nitrile 4 25 175 <![CDATA[NCH3]]> <![CDATA[CH2CH2CH2CH2CH2]]> Thiol 14 30 176 <![CDATA[NCH3]]> <![CDATA[CH2CH2]]> Maleimide 4 35 177 CONH <![CDATA[CH2CH2CH2]]> Epoxy 4 25 178 CONH <![CDATA[CH2CH2CH2CH2]]> Aziridine 4 30 179 CONH <![CDATA[CH2]]> Ethyl sulfide 4 35 180 CONH <![CDATA[CH2CH2CH2CH2CH2CH2]]> Triazolyl 4 30

[0160] In the above Table 6, P in compound numbers 151 to 160 represents CH2CH2C * H(NH2)CH2 (here, the asymmetric carbon is R type), P in compound numbers 161 to 170 represents CH2CH2C * H(NH2) (here, the asymmetric carbon is S-type), and P in compound numbers 171 to 180 represents CH2C *H(NH2) (here, the asymmetric carbon is R-type). As the pharmacologically acceptable salt of the general formula (1) or (4), for example, salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, for example, salts with organic acids such as oxalic acid, maleic acid, succinic acid, acetic acid, for example, salts with inorganic bases such as sodium and potassium, for example, salts with organic bases such as dimethylamine and triethylamine.

[0161] The compound represented by the general formula (1) or a salt thereof can be reacted with the general formula (6)

[0162]

[0163] (Where P + Indicates that 1 or 2 or more (preferably 1) N + It can be produced by subjecting a compound represented by the general formula HQZ (wherein, Q and Z have the same meanings as above) or a salt thereof to a condensation reaction.

[0164] In addition, the compound represented by the general formula (4) or a salt thereof can be reacted with the general formula (7)

[0165]

[0166] (In the formula, n has the same meaning as above.)

[0167] The ε-PL and general formula (3a) shown

[0168]

[0169] (Wherein, X, Y and m have the same meanings as above.)

[0170] The compound shown or a salt thereof can be produced by subjecting the compound to a condensation reaction.

[0171] Before the condensation reaction, the amino group involved in the reaction of the raw material can be pre-protected by a protecting group, which is removed after the condensation reaction, thereby obtaining a compound represented by the general formula (1) or a salt thereof as the target. The method of condensation reaction has been well established in this field so far, and therefore, the present invention can proceed accordingly. The compound represented by the general formula (1) can be obtained by conventional means such as concentration, dissolution, chromatography, etc. of the synthetic reaction solution of the target. Such technology has been well established in the field of peptide synthesis, and therefore, the present invention can proceed accordingly. The compound represented by the general formula (6) (including the compound ε-PL represented by the general formula (7)) can be obtained by any method such as fermentation, chemical synthesis, or other chemical reactions, or can be obtained by chemical synthesis from lysine. For example, ε-PL can be obtained by culturing Streptomyces Alblas Subspecies Lysinopolymeras described in Japanese Patent No. 1245361 in a culture medium, for example, a culture medium adjusted to 5 wt% glucose, 0.5 wt% yeast extract, 1 wt% ammonium sulfate, 0.08 wt% dipotassium hydrogen phosphate, 0.136 wt% potassium dihydrogen phosphate, 0.05 wt% magnesium sulfate heptahydrate, 0.004 wt% zinc sulfate heptahydrate, 0.03 wt% iron sulfate heptahydrate, and pH 6.8, and isolating and collecting ε-polylysine from the obtained culture.

[0172] In addition, the polycationic body including the analog of the compound ε-PL represented by the general formula (7) can be obtained by any method, and can also be obtained by the method described in the following non-patent literature.

[0173] For example, γ-poly-L-diaminobutyric acid can be produced by referring to the paper (Takehara, M., Saimura, M., Inaba, H. & Hirohara, H. Poly (gamma-L-diaminobutanoic acid), a novel poly (aminoacid), coproduced with poly (epsilon-L-lysine) by two strains of Streptomyces celluloflavus. FEMS Microbiol Lett 286, 110-7 (2008).).

[0174] In addition, γ-poly-D-diaminobutyric acid can be produced by referring to a paper (Ohkuma, H., Tenmyo, O., Konishi, M., Oki, T. & Kawaguchi, H. BMY-28190, a novel antiviral antibiotic complex. J Antibiot (Tokyo) 41, 849-54 (1988).), for example.

[0175] In addition, β-poly-L-diaminopropionic acid can be produced by referring to the papers (Xia, J., Xu, H., Feng, X., Xu, Z. & Chi, B. Poly (L-diaminopropionic acid), a novel non-proteinic amino acid oligomer co-produced with poly (epsilon-L-lysine) by Streptomyces albulus PD-1. Appl Microbiol Biotechnol 97, 7597-605 (2013). or Xu, Z. et al. Systematic unravelling of the biosynthesis of poly (L-diaminopropionic acid) in Streptomyces albulus PD-1. Sci Rep 5, 17400 (2015).).

[0176] The polycations such as ε-PL represented by the general formula (7) used in the present invention can also be used as salts. Examples of such salts include inorganic acid salts of polycations formed by inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid and polycations, organic acid salts of polycations formed by organic acids such as acetic acid, formic acid, propionic acid, fumaric acid, malic acid, citric acid, maleic acid, adipic acid, gluconic acid and lactic acid and polycations, saturated fatty acid salts of polycations formed by medium-chain and long-chain saturated fatty acids such as caproic acid, lauric acid and stearic acid and polycations, and unsaturated fatty acid salts of polycations formed by medium-chain and long-chain unsaturated fatty acids such as oleic acid, linoleic acid and arachidonic acid and polycations.

[0177] The compounds represented by the general formulae (2a) and (3a) are known compounds or compounds similar to known compounds. Therefore, the compounds represented by the general formulae (2a) and (3a) can be easily produced by known methods or methods known per se.

[0178] The compound represented by the general formula (1) or (4) can be produced simply and efficiently by the following microbial culture method.

[0179] The microorganism used in the present invention may be any microorganism as long as it has the ability to produce polycations such as ε-PL, and preferably is a bacterium of the genus Kitasatospora or a bacterium of the genus Streptomyces.

[0180] Examples of the Kitasatospora bacteria include those described in International Journal of Systematic Bacteriology, Vol. 47, pp. 1048-1054, 1997. Among them, Kitasatospora akifunense is preferred, and Kitasatospora Kifunense MN-1 strain (FERMP-19712) is more preferred.

[0181] Examples of the genus Streptomyces include Streptomyces albulus subsp. lysinopolymerus No. 346-D strain (IFO14147, Japanese Patent Publication No. 59-20359), Streptomyces albras subspecies lysinopolymeras 11011A strain (Japanese Patent Publication No. 3-42070), Streptomyces albras subspecies lysinopolymeras B21021 strain (Japanese Patent Application Publication No. 09-173057), Streptomyces albras subspecies lysinopolymeras B15208 strain (Japanese Patent Application Publication No. 10-290688), Streptomyces Albras Subspecies SP-25 strain (Japanese Patent Application Laid-Open No. 2002-095467), Streptomyces sp. SP-72 strain (Japanese Patent Application Laid-Open No. 2000-069988), Streptomyces sp. SP-66 strain (Japanese Patent Application Laid-Open No. 2001-017159), Streptomyces herbali color SP-13 strain (Japanese Patent Application Laid-Open No. 2002-095466), Streptomyces Ravendurae USE-53 strain (Japanese Patent Application Laid-Open No. 2003-052358), Streptomyces norsei (Japanese Patent Application Laid-Open No. 1-187090), etc. Among them, microorganisms classified into Streptomyces albus are preferred, and Streptomyces albras subspecies lysinopolymeras No. 346-D strain (the same strain as Streptomyces albulus NBRC14147 strain) is more preferred.

[0182] Furthermore, the microorganism used in the present invention may be a microorganism that produces analogs of ε-PL such as the above-mentioned γ-poly-L-diaminobutyric acid, γ-poly-D-diaminobutyric acid, and β-poly-L-diaminopropionic acid.

[0183] The compound represented by the general formula (2a) or (3a) including a click functional group is added to the culture medium for culturing these microorganisms. The compound represented by the general formula (2a) or (3a) can be any compound as long as it falls within the scope of the compound represented by the general formula (2a) or (3a), and examples thereof include PEG-azide, PEG-alkyne (i.e., alkynyl PEG), etc. The concentration of the compound represented by the general formula (2a) or (3a) added to the culture medium is preferably 0.1% to 1.0% (% is volume percentage). In addition, as a culture medium component other than the compound represented by the general formula (2a) or (3a), any culture medium can be used as long as it appropriately contains a carbon source, a nitrogen source, an inorganic substance, and other nutrients. As a carbon source, any carbon source can be used as long as it can be assimilated by the polycation producing strain, and examples thereof include glucose, fructose, starch, glycerol, etc. The content of the carbon source is preferably 1.0% to 10.0%, and more preferably 1.0% to 5.0%. As the nitrogen source, for example, peptone, casein hydrolyzate, amino acid, inorganic ammonium salt, ammonium sulfate, etc. can be mentioned, among which ammonium sulfate is preferred. The content of the nitrogen source is preferably 0.1% to 5.0%, and more preferably 0.1% to 1.0%. The carbon source or nitrogen source can be added sequentially during the culture. As inorganic substances, phosphate ion, potassium ion, sodium ion, magnesium ion, zinc ion, iron ion, magnesium ion, nickel ion, sulfate ion, etc. can be mentioned. In order to make the growth of bacteria good, it is preferred that yeast extract is contained in the culture medium. The content of yeast extract is preferably 0.05% to 0.5%, and more preferably 0.1% to 0.5%. The pH of the culture medium can be any pH that can culture microorganisms, for example, pH 3 to pH 8 can be mentioned, among which neutrality (pH 6 to 8) is preferred for the growth of microorganisms, and pH 3 to pH 5 is more preferred for the production of the compound represented by the general formula (1). In addition, in the present invention, % is w / v% unless otherwise specified. For more details, refer to the following examples.

[0184] The culture is preferably carried out under aerobic conditions with oxygen supply, such as shaking culture or stirring culture. The culture temperature is not particularly limited as long as the polycation producing strain can grow, and examples thereof include 25 to 35° C. After the start of the culture, the pH of the culture solution decreases as the bacteria proliferate, but as a method for maintaining the pH, for example, an alkaline solution is added to the culture solution, and a method of maintaining the pH of the culture solution at about 4 can be cited. The alkaline solution added is not particularly limited, and for example, sodium hydroxide, ammonia water, etc. can be cited.

[0185] After centrifugation or filtration of the above-mentioned culture solution to remove the bacterial cells, the bacterial cell removal solution is adsorbed on a weakly acidic cation exchange resin. The weakly acidic cation exchange resin is not particularly limited, and for example, Amberlite IRC-50 (manufactured by Rohm and Haas Company) can be cited. After adsorption, the polycationic body derivative shown in the general formula (1) can be obtained by dissolving with hydrochloric acid. In addition, it can also be separated and purified by the tetraphenyl borate method (non-patent literature, H.Katano, etc., Anal.Sci., 28, 1153-1157, 2012).

[0186] Next, the compound represented by the general formula (1) is introduced into the pharmacological substance through a covalent bond using click chemistry. Click chemistry is well known to those skilled in the art. In the present invention, such known techniques can be used. As the pharmacological substance used in the present invention, any substance can be used as long as the compound represented by the general formula (1) or its salt can be introduced by covalent bond formation based on the click functional group. For example, a compound having a triple bond (e.g., -C≡C-) is preferred. In short, as the pharmacological substance, a substance such as a pharmacological substance that is chemically bonded to the click functional group reaction is preferred. Thus, biomembrane permeability and water solubility are improved at the same time.

[0187] As the pharmacologically active substance, for example, antibacterial agents containing antifungal agents, antibacterial antibiotics, anticancer agents, proteins such as enzymes or antibodies, genes, etc. As antifungal agents, for example, polyene antibiotics (amphotericin B, nystatin, aspergillus, pimazine, etc.), micafungin, itraconazole, ketoconazole, fluconazole, miconazole, flucytosine, etc. As antibacterial antibiotics, for example, β-lactam antibiotics (penicillin, ampicillin, talancillin, bacillin, cephalosporin C, cephalexin, cephradine, cephamycin, imipenem, etc.), aminoglycoside antibiotics (streptomycin, gentamicin, kanamycin, tobramycin, amikacin, arbekacin, nucleomycin, etc.), tetracycline antibiotics (oxytetracycline, tetracycline, etc.), peptide antibiotics (bacitracin, gramicidin, polymyxin B, vancomycin, teicoplanin, etc.), etc. As anticancer agents, for example, anticancer agents capable of introducing click functional groups, etc., can be mentioned. As anticancer agents capable of introducing click functional groups, for example, actinomycin D, mitomycin, anthracycline antitumor drugs (doxorubicin, daunorubicin, aclarubicin, etc.), bleomycin, cisplatin can be mentioned. As proteins, fluorescent proteins such as mAG, Nakayama factor proteins as gene products of Cas proteins (Cas, Cas9) used in the CRISPR-Cas9 system, Oct3 / 4, Sox2, Klf4 and c-Myc, gene expression control proteins, etc. can be exemplified. As enzymes, trypsin, CreRecombinase, λ-Redrecombinase, etc. As antibodies, immunoglobulins such as IgG antibodies, IgA antibodies, and IgY antibodies can be exemplified. As genes, plasmids, oligonucleotides, Nakayama factors such as Oct3 / 4, Sox2, Klf4 and c-Myc, PAM sequences used in the CRISPR-Cas9 system, gRNA (guide RNA, guideRNA, sgRNA), etc. are used.

[0188] When the compound represented by the general formula (1) or its salt is introduced into the pharmacological substance, it is convenient to use an insertion group used as a common means in the field of click chemistry. In short, the insertion group is a group that can be inserted to integrate the pharmacological substance and the compound represented by the general formula (1) or its salt. Any group can be used as long as it does not hinder the purpose of the present invention. As such an insertion group, for example, an amino group, carboxyl group or hydroxyl group of a compound originally having an insertion group reacts with a carboxyl group, hydroxyl group or amino group in the pharmacological substance to form a covalent bond, and the triple bond in the compound reacts with the click functional group in the compound represented by the general formula (1) or its salt to form a covalent bond. The types of such groups and the methods of introduction into the pharmacological substance (amidation reaction, esterification reaction, Huisgen reaction, etc.) have been fully established in the field of click chemistry, so the present invention can be carried out accordingly.

[0189]

[0190] When introducing the compound represented by the general formula (1) or a salt thereof into a pharmacological substance, for example, a click chemistry insertion compound such as DBCO (dibenzocyclooctyne) may be inserted. That is, it is advantageous to introduce the compound represented by the general formula (1) or a salt thereof into a compound obtained by combining a pharmacological substance with a click chemistry insertion compound such as DBCO.

[0191] For example, examples of introducing a pharmacologically active substance into a compound represented by the general formula (1) via a covalent bond include the following.

[0192] By using formula (8)

[0193]

[0194] The poorly water-soluble Amp-B shown in the figure is used to carry out an organic synthesis reaction to produce formula (9):

[0195]

[0196] The reaction can be carried out by a known method or a known amidation reaction. The compound (9) or a salt thereof prepared in this way is reacted with the general formula (10):

[0197]

[0198] (wherein n is an integer from 2 to 100) or a salt thereof to produce the general formula (5):

[0199]

[0200] (wherein n is an integer from 2 to 100) is a water-soluble ε-PL-DBCO-AmpB or a salt thereof. For more details, refer to the following Examples.

[0201] The water-soluble ε-PL-DBCO-AmpB or its salt represented by the general formula (5) thus produced can maintain the antibacterial efficacy of amphotericin B, which is useful as a drug for the treatment of deep fungal diseases, while overcoming the disadvantages of amphotericin B, namely, difficulty in intestinal absorption, difficulty in oral administration, and dependence on intravenous administration, and also overcomes the disadvantages of poor water solubility (long intravenous infusion time).

[0202] The water-soluble ε-PL-DBCO-Dox represented by the general formula (5-2) and its salt can be produced by the same method as the method for producing the compound represented by the general formula (5). For more details, refer to the following Examples.

[0203] The water-soluble ε-PL-DBCO-Dox or its salt represented by the general formula (5-2) thus produced can not only maintain the anticancer activity of doxorubicin, which is useful as an anticancer agent, but also reduce intestinal absorption and side effects. The Dox structure of ε-PL-DBCO-Dox is embedded in the double helix structure of DNA, so only by mixing plasmid DNA, gene fragments, or RNA with ε-PL-DBCO-Dox, polycation modification can be performed without covalently bonding DNA or RNA. By this method, polycation-modified plasmid DNA or gene fragments, or RNA can be directly delivered into cells. For more details, refer to the following examples.

[0204] ε-PL-DBCO-mAG represented by the general formula (5-3) or a salt thereof can be produced by the same method as the method for producing the compound represented by the general formula (5). For more details, refer to the following Examples.

[0205] The ε-PL-DBCO-mAG or its salt represented by the general formula (5-3) thus produced can directly deliver mAG as a fluorescent protein into cells. Similarly, high molecular weight pharmacological substances, such as proteins (including enzymes and antibodies), can also be directly delivered into cells by modifying them with ε-PL.

[0206] The polycation introduction method of the click functional group of the present invention can also be applied to, for example, industrial substrates (for example, medical substrates or functional materials such as plastic products or fibers). As medical substrates, for example, cell culture dishes, medical instruments, regenerative medical substrates, etc. can be cited. As functional materials, for example, electronic substrates, special coated glass, etc. can be cited. By introducing the polycation of the present invention into industrial substrates, etc., for example, antibacterial activity, cultured cell adhesion effect, hydrophilic effect, anti-drip effect, antistatic effect, etc. can be given.

[0207] Several embodiments are shown below to further explain the implementation of the present invention in detail. The present invention is not limited to the following embodiments, and there may be various ways to the details. In addition, the present invention is not limited to the implementation of the technology, and various changes shown in the claims can be made, and the implementation obtained by appropriately combining the disclosed technical means is also included in the technical scope of the present invention.

[0208] [Example 1]

[0209] Method for producing a compound represented by general formula (1) by microorganisms

[0210] Streptomyces albulus NBRC1417 was used as the ε-PL producing bacterium, and plsL883P / pLAE009 / S. albulusCRM003 was used as the short-chain ε-PL producing bacterium (Non-patent literature: Y. Hamano et al., Appl. Environ. Microbiol., 80, 4993-5000, 2014). In addition, sterile SLB medium (a substance in which each component of sucrose, 10.3 g; tryptone, 10 g; and yeast extract, 5 g, was dissolved in 1 L of distilled water) and M3G medium (a substance in which each component of glucose, 50 g; ammonium sulfate, 10 g; yeast extract, 5 g; potassium dihydrogen phosphate, 1.36 g; sodium hydrogen phosphate dodecahydrate, 1.58 g; zinc sulfate heptahydrate, 0.04 g; iron (II) sulfate heptahydrate, 0.03 g; and magnesium sulfate heptahydrate, 0.5 g, was dissolved in 1 L of distilled water and adjusted to pH 7.0 with an aqueous sodium hydroxide solution) were used for the culture.

[0211] A spore suspension of Streptomyces albulusNBRC14147 or plsL883P / pLAE009 / S.albulusCRM003 was inoculated into 3 mL of SLB medium and cultured with shaking at 28°C for 36 hours. In addition, in the culture of plsL883P / pLAE009 / S.albulusCRM003, the antibiotic neomycin was added to the medium at a concentration of 50 μg / mL as a selective agent. 500 μL of the culture solution was added to 50 mL of M3G medium, and the mixture was cultured at 28° C. for 8 hours. As the compound represented by the general formula (2a) or (3a), 2-[2-[2-(2-propyn-1-yloxy)ethoxy]ethoxy]ethanol (hereinafter referred to as PEG-alkyne) or 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethanol (hereinafter referred to as PEG-azide) was added to the culture solution to a concentration of 0.2% (w / v), and the mixture was further cultured with shaking at 28° C. for 2 to 4 days. The culture solution was centrifuged to obtain a culture supernatant.

[0212] The structure of general formula (1) is determined

[0213] The culture supernatant of Streptomyces albulus NBRC14147 obtained in Example 1 was analyzed by electrospray ionization time-of-flight mass spectrometry / high performance liquid chromatography (hereinafter referred to as ESI-TOF-MS / HPLC) to determine the precise molecular weight ( Figure 1A and 1B). The ESI-TOF-MS using ESI-TOF-MS / HPLC used maXisplus manufactured by Bruker Daltonics, and the HPLC used Agilent 1290 Infinity LC system manufactured by Agilent Technologies. The HPLC conditions were as follows: the chromatographic column was SunShell RP-AQUA (manufactured by Chromanik technology, 2.6 μm, inner diameter 2.1 mm, length 50 mm), the column temperature was 40°C, the mobile phase A used an aqueous solution containing 0.05% formic acid and 0.05% heptafluorobutyric acid, and the mobile phase B used acetonitrile containing 0.05% formic acid and 0.05% heptafluorobutyric acid. The mobile phase flow rate was set to 0.3 mL / min, and 3 minutes before the start of the analysis, a linear gradient of 10% to 25% of mobile phase B was used, a linear gradient of 25% to 90% of mobile phase B was used from 3 minutes to 12 minutes, a linear gradient of 90% to 100% of mobile phase B was used from 12 minutes to 16 minutes, and a mobile phase B of 100% was used from 16 minutes to 18 minutes. The monitoring of the elution was carried out by ESI-TOF-MS measurement.

[0214] result

[0215] It was confirmed that the compound represented by the general formula (1) in which the carboxyl group of ε-PL and PEG-azide were ester-bonded, i.e., the ε-PL-PEG-azide represented by the general formula (10) (wherein n is an integer from 17 to 26), and the compound represented by the general formula (1) in which the carboxyl group of ε-PL and PEG-alkyne were ester-bonded, i.e., the ε-PL-PEG-alkyne represented by the general formula (11) (wherein n is an integer from 17 to 26) were produced. In addition, ε-PL-PEG-azide and ε-PL-PEG-alkyne were separated and purified from the culture medium by the tetraphenyl borate method (non-patent literature H. Katano et al., Anal. Sci., 28, 1153-1157, 2012), and their chemical structures were confirmed by nuclear magnetic resonance spectroscopy (1H-NMR) ( Figure 2A and 2B ). Based on the results, it was determined that ε-PL-PEG-azide and ε-PL-PEG-alkyne composed of 17-26mer ε-PL were produced by using Streptomyces albulus NBRC14147 as an ε-PL producing bacterium. On the other hand, by using plsL883P / pLAE009 / S.albulusCRM003 as a short-chain ε-PL producing bacterium, short-chain ε-PL-PEG-azide and ε-PL-PEG-alkyne could be produced.

[0216]

[0217] [Example 2]

[0218] Simultaneous improvement of water solubility and biomembrane permeability through ε-PL modification of compounds

[0219] As a model experiment for simultaneously improving the water solubility and biomembrane permeability of a compound through ε-PL modification, ε-PL modification of a poorly water-soluble fluorescent dye (DBCO-PEG4-5 / 6-FAM) represented by formula (12) was performed.

[0220]

[0221] Commercially available DBCO-PEG4-5 / 6-FAM (0.5 μmol) represented by formula (12) and ε-PL-PEG-azide (1.0 μmol) composed of 9-18mer ε-PL represented by general formula (10) (wherein n is an integer from 9 to 18) were dissolved in 80% dimethyl sulfoxide (hereinafter referred to as DMSO), and click chemistry reaction was carried out at 30°C for 24 hours while stirring. After analyzing the reaction solution by ESI-TOF-MS / HPLC according to the method described in Example 1, it was confirmed that all the increased DBCO-PEG4-5 / 6-FAM was converted into the compound (ε-PL-DBCO-FAM) represented by general formula (13) (wherein n is an integer from 9 to 18) ( Figure 3A and 3B ). In addition, it was found that ε-PL-DBCO-FAM purified from the reaction solution was easily soluble in water, and that DBCO-PEG4-5 / 6-FAM, which had poor water solubility, was modified with ε-PL to be water soluble.

[0222]

[0223] DBCO-PEG4-5 / 6-FAM represented by formula (12) did not show biomembrane permeability. Here, HeLa cells as animal cells were used to confirm the biomembrane permeability of ε-PL-modified DBCO-PEG4-5 / 6-FAM, i.e., ε-PL-DBCO-FAM represented by general formula (13) (wherein n is an integer from 9 to 18). In the culture of HeLa cells, Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) was used, and cultured at 37°C in an atmosphere containing 5% carbon dioxide. Under this culture condition, HeLa cells were cultured in a 12-well plate until 70-80% confluence, and the cells were washed twice with DMEM without FBS (DMEM w / o FBS). DMEM w / o FBS was added to the cells, and after cooling to 4°C, DBCO-PEG4-5 / 6-FAM represented by formula (12) or ε-PL-DBCO-FAM represented by general formula (13) (wherein n is an integer from 9 to 18) was added to a concentration of 30 μM, respectively, and incubated at 4°C for 30 minutes. The cells were washed twice with phosphate-buffered saline (PBS), washed with PBS containing heparin (100 μg / mL) or with PBS alone, and then washed twice with PBS. 300 μL of 100 mM Tris-HCl buffer (pH 8.0) containing 1% Triton X-100 was added to extract the compounds that entered the cells, and after removing the insoluble portion by centrifugation, the fluorescence intensity was measured. As a result, it was confirmed that the biomembrane permeability of ε-PL-DBCO-FAM represented by general formula (13) was about 3.5 times higher than that of DBCO-PEG4-5 / 6-FAM represented by formula (12) in both the case where heparin washing was performed and the case where heparin washing was not performed to wash away the ε-PL-DBCO-FAM bound to the cell surface. Figure 4 ). In addition, it was demonstrated that when heparin was not washed, since the fluorescence of ε-PL-DBCO-FAM was highly elevated during treatment, part of ε-PL-DBCO-FAM bound to the cell surface was taken up into the cell ( Figure 4 In fact, it was confirmed by confocal laser microscopy that ε-PL-DBCO-FAM represented by the general formula (13) existed in the cell, and part of it was also present in the nucleus ( Figure 5 ).

[0224] This experiment revealed that the modification of compounds with ε-PL can simultaneously achieve the goals of imparting water solubility and improving biomembrane permeability.

[0225] [Example 3]

[0226] In order to further evaluate the usefulness of the compound represented by the general formula (1), i.e., the ε-PL-PEG-azide represented by the general formula (10) (wherein n is an integer from 9 to 18), the antifungal antibiotic AmpB represented by the formula (8) was modified by ε-PL. AmpB is an important clinical antibiotic used as a therapeutic drug for various fungal infections represented by deep fungal diseases because it exhibits antibacterial activity against fungi. However, since it is basically insoluble in water and has poor water solubility, it has problems such as complicated formulation adjustment and side effects such as nephrotoxicity. In addition, various formulation technologies such as liposome formulations to improve its poor water solubility have been discussed, but no completely satisfactory results have been obtained. AmpB is basically not absorbed by the intestine after oral administration, and therefore, the problem of intestinal absorption cannot be overcome in the latest formulation technology. On the other hand, if ε-PL-modified AmpB can maintain strong antifungal activity, it is related to the development of AmpB that is easily soluble in water, and it is expected to improve the above-mentioned multiple problems at the same time.

[0227] ε-PL modification of the antifungal antibiotic AmpB

[0228] AmpB (manufactured by Nakarai Tesque Co., Ltd.) 100 mg and N-[(9H-fluoro-9-methoxy)carbonyloxy]succinimide (Fmoc-OSu) 73 mg were dissolved in anhydrous dimethylformamide 30 mL. The mixture was stirred at 28°C for 4 hours to synthesize a compound (AmpB-Fmoc) in which the amino group of the amino sugar of AmpB was protected by an Fmoc group. 420 mL of 25 mM hydrochloric acid aqueous solution was added to the reaction solution, and the white precipitate AmpB-Fmoc obtained by centrifugation was dissolved in DMSO and freeze-dried to obtain a powder of AmpB-Fmoc. AmpB-Fmoc 24.8 mg, N,N-diisopropylethylamine 8.88 μL, and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride 9.0 mg were dissolved in anhydrous dimethylformamide 4.5 mL. After the reaction solution was stirred at room temperature for 30 minutes, 12.5 mg of commercially available DBCO-PEG4-amine was added, and the mixture was stirred at 28°C for 5 hours. 1.2 mL of piperidine was added to deprotect the Fmoc group, and AmpB-DBCO represented by general formula (9) was synthesized. 120 mL of diethyl ether was added to the reaction solution, and the yellow precipitate AmpB-DBCO obtained by centrifugation was dissolved in DMSO and purified by high performance liquid chromatography (HPLC) using a reverse phase column (Sunniest RP-AQUA, 5 μm, 250×10 mm, ChromaNik Technologies). The HPLC conditions are as follows. Column temperature, 30°C; detection, absorption at UV405 nm; mobile phase A, 0.1% (v / v) formic acid aqueous solution; mobile phase B, acetonitrile; mobile phase gradient, linear gradient of 40% to 100% of B solution over 30 minutes. Acetonitrile was distilled off from the eluted component containing AmpB-DBCO represented by general formula (9), and the mixture was freeze-dried to obtain a yellow powder of AmpB-DBCO represented by general formula (6). 366 μg of AmpB-DBCO represented by general formula (9) and 689 μg of ε-PL-PEG-azide composed of 9-18mer ε-PL represented by general formula (7) were dissolved in 86% DMSO aqueous solution, and click chemistry reaction was carried out at 30°C for 5 hours while stirring. The reaction solution was analyzed by ESI-TOF-MS / HPLC, confirming the synthesis of ε-PL-DBCO-AmpB represented by general formula (5) (wherein n is an integer from 9 to 17) ( Fig. 6A and 6B ).

[0229] Water solubility of ε-PL-DBCO-AmpB represented by general formula (5)

[0230] When ε-PL-DBCO-AmpB represented by the general formula (5) (wherein n is an integer from 9 to 17) and AmpB represented by the formula (8) were dissolved in water, AmpB was completely insoluble in water at a concentration of 1.08 mM, but ε-PL-DBCO-AmpB represented by the general formula (5) (wherein n is an integer from 9 to 17) was easily soluble in water at a concentration of 5.63 mM, showing high water solubility ( Figure 7 ). Thus, it can be seen that ε-PL modification of poorly water-soluble AmpB can impart high water solubility, and it is shown that by using the ε-PL-PEG-azide represented by the general formula (10) (wherein n is an integer from 9 to 18), drugs such as pharmaceuticals can be effectively and simply modified with ε-PL, which clarifies its usefulness.

[0231] Antifungal activity of ε-PL-DBCO-AmpB represented by general formula (5)

[0232] Aspergillus brasiliensis NBRC9455 was used as a test fungus, and the minimum growth inhibitory concentration (MIC) of ε-PL-DBCO-AmpB represented by the general formula (5) (wherein n is an integer from 9 to 17), AmpB represented by the formula (8), and ε-PL-PEG-azide composed of 9-18mer ε-PL represented by the general formula (10) (wherein n is an integer from 9 to 18) was measured. The commercially available RPMI-1640 medium was used as the culture medium, and when cultured at 30°C for 5 days, the MIC of ε-PL-DBCO-AmpB represented by the general formula (5) was 1.9 μg / mL, the MIC of AmpB represented by the formula (8) was 0.3 μg / mL, and the MIC of ε-PL-PEG-azide composed of 9-18mer ε-PL represented by the general formula (10) was 72 μg / mL. From the above results, it can be seen that ε-PL modified AmpB, i.e. ε-PL-DBCO-AmpB shown in general formula (5), is water-soluble and maintains sufficient antifungal activity. The poorly water-soluble AmpB shown in formula (8) is not absorbed by the intestines after oral administration and is therefore only suitable for fungal infections of the digestive tract such as the oral cavity or esophagus. However, it can be seen that the water-soluble ε-PL-DBCO-AmpB shown in general formula (5) can improve biomembrane permeability through ε-PL modification (polycation modification), and can be used for deep fungal diseases even if it is orally administered.

[0233] [Example 4]

[0234] ε-PL modification of the anticancer agent Dox

[0235] 17.4 mg of Dox (hydrochloride, manufactured by Funakoshi Co., Ltd.), 19.4 mg of DBCO-PEG4-NHSEster and 4.9 mg of N,N-dimethyl-4-aminopyridine were dissolved in 3 mL of DMSO. Stirring was performed at room temperature for 4 hours to synthesize a compound (DBCO-Dox) in which the amino group of the amino sugar of Dox was bound to DBCO-PEG4. The synthesized DBCO-Dox was purified by high performance liquid chromatography (HPLC) using a reverse phase chromatography column (Sunniest RP-AQUA, 5 μm, 250×10 mm, ChromaNik Technologies). The HPLC conditions are as follows. Column temperature, 35°C; detection, UV254nm absorption; mobile phase A, 0.05% (v / v) formic acid aqueous solution and 0.05% (v / v) HFBA aqueous solution; mobile phase B, 0.05% (v / v) formic acid acetonitrile solution and 0.05% (v / v) HFBA acetonitrile solution; mobile phase gradient elution, 18 minutes 50% isocratic elution of liquid B. Acetonitrile was distilled off from the eluted component containing DBCO-Dox, DBCO-Dox was extracted with ethyl acetate and dried. DBCO-Dox was completely dissolved in 1 mL of chloroform and dried again. 10 μmol of DBCO-Dox and 11 μmol of ε-PL-PEG-azide were dissolved in 80% DMSO aqueous solution, and the click chemistry reaction was generally carried out for 3 hours at room temperature while stirring. The reaction solution was analyzed by ESI-TOF-MS / HPLC to confirm that ε-PL-DBCO-Dox represented by the general formula (5-2) (wherein n is an integer from 18 to 29) was synthesized ( Fig. 8A and B).

[0236] Cytotoxicity (anticancer activity) of ε-PL-DBCO-Dox represented by general formula (5-2)

[0237]

[0238] Regarding Dox represented by formula (14) and ε-PL-DBCO-Dox represented by general formula (5-2) (wherein n is an integer from 18 to 29), the cytotoxicity against human chronic myeloid leukemia cells K562 cells and Dox-resistant K562 cells K562 / ADR cells was verified. K562 cells and K562 / ADR cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) at 37°C in an atmosphere containing 5% carbon dioxide. Each cell was suspended in the culture medium to a concentration of 1×10 5 The concentration of cells / mL is 1×10 4Cells) were seeded on 96-well plates and cultured for 24 hours. 100 μL of culture medium containing various concentrations of Dox and ε-PL-DBCO-Dox was added and cultured for 48 hours. The number of viable cells was measured by MTT assay and the cytotoxicity was calculated. The results showed that the IC of Dox in K562 cells and K562 / ADR cells was 50 The IC values ​​of ε-PL-DBCO-Dox in K562 cells and K562 / ADR cells were 239±72nM and 1,043±72nM, respectively. 50 They are 12.5±1.31μM and 49.9±9.43μM, respectively. Therefore, although it is confirmed that the anticancer activity of ε-PL-DBCO-Dox represented by general formula (5-2) is reduced by about 50 times compared with Dox represented by formula (14), while maintaining the anticancer activity of doxorubicin, which is useful as an anticancer agent, the absorption and side effects from the intestine can be reduced. In addition, since the present results show that the Dox structure of ε-PL-DBCO-Dox is embedded in the double helix structure of DNA, polycation modification can be performed without covalently bonding DNA or RNA simply by mixing plasmid DNA or gene fragments, or RNA with ε-PL-DBCO-Dox. In this method, polycation-modified plasmid DNA or gene fragments, or RNA can be directly delivered into cells.

[0239] [Example 5]

[0240] ε-PL modification of macromolecular pharmacological substances

[0241] As an example of a high molecular weight pharmacological substance, the monomeric Azami-Green (mAG) fluorescent protein was modified with ε-PL. A plasmid (mAG / pET21a) for highly expressing mAG as a C-terminal his tag fusion protein was introduced into Escherichia coli BL21 (DE3). The E. coli was inoculated in LB medium (containing 100 μg / mL of ampicillin), cultured at 37°C for 2.5 hours, induced with 0.1 mM IPTG for mAG expression, and then cultured overnight at 18°C. The cells were recovered, suspended in a buffer solution (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 7.0), and then disrupted by ultrasound. The disrupted liquid was centrifuged to recover the cell-free extract, and mAG was purified using a Ni affinity chromatography column. The purified mAG was dialyzed overnight using a dialysis external solution of 100 mM sodium bicarbonate buffer (pH 9.0). To mAG, 1 equivalent of DBCO-sulfo-NHS was added three times within 1 hour at 4°C while stirring, and the reaction was carried out for a total of 3 hours to prepare DBCO-modified mAG (DBCO-mAG). After the dialyzed external fluid was dialyzed overnight with 100 mM sodium phosphate buffer (pH 6.0), 1.1 equivalents of ε-PL-PEG-azide composed of 20-30mer ε-PL represented by general formula (10) (wherein n is an integer from 20 to 30) was added to DBCO-mAG, and the reaction was carried out at 4°C for 1 hour. After the reaction solution was analyzed by ESI-TOF-MS / HPLC, it was confirmed that ε-PL-DBCO-mAG ( Fig.9A , 9B and 9C).

[0242] Evaluation of biomembrane permeability of ε-PL-DBCO-mAG

[0243] Generally, proteins cannot pass through biological membranes such as cell membranes. Therefore, animal cells HeLa cells were used to confirm whether ε-PL-modified mAG, that is, ε-PL-DBCO-mAG represented by general formula (5-3) (wherein n is an integer from 23 to 29), can pass through biological membranes. HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C in an atmosphere containing 5% carbon dioxide. Under these culture conditions, HeLa cells were cultured on a 12-well plate until they reached 70-80% confluence, and the cells were washed twice with DMEM without FBS (DMEMw / oFBS). DMEMw / oFBS was added to the cells, and after cooling to 4°C, mAG or ε-PL-DBCO-mAG represented by general formula (5-3) was added to 50 μM, respectively, and incubated at 4°C for 30 minutes. The cells were washed twice with phosphate-buffered saline (PBS), washed with PBS containing heparin (100 μg / mL), and then washed twice with PBS. 100 mM Tris-HCl buffer (pH 8.0) containing 300 μL of 1% Triton X-100 was added to extract the compounds taken into the cells, and the insoluble components were removed by centrifugation, and the fluorescence intensity was measured. As a result, it was confirmed that the biomembrane permeability of ε-PL-DBCO-mAG represented by general formula (5-3) was about 10 times higher than that of mAG represented by general formula (5-3) ( Fig.10 In fact, observation with a confocal laser microscope visually confirmed that ε-PL-DBCO-mAG represented by the general formula (5-3) was present in the cell, and part of it was also present in the nucleus ( Fig.11 ).

[0244] [Example 6]

[0245] Chemical stability test of ε-PL-PEG-alkyne represented by general formula (11)

[0246] The chemical stability of the ester bond of the ε-PL-PEG-alkyne represented by the general formula (11) (wherein n is an integer from 17 to 26) obtained by the same method as in Example 1 and the ε-PL-alkyne represented by the general formula (15) (wherein n is an integer from 17 to 26) obtained by the same method as in Example 1 was verified. The ε-PL-PEG-alkyne and the ε-PL-alkyne were dissolved in a Tris-HCl buffer (pH 8.0) at a concentration of 1 mM, heated at 60°C for 5 minutes, and analyzed by ESI-TOF-MS / HPLC. As a result, it was found that the ester bond of the ε-PL-PEG-alkyne is difficult to hydrolyze and has high chemical stability ( Fig. 12A and B).

[0247]

[0248] Industrial Applicability

[0249] The present invention provides not only a polycationic derivative that improves the biomembrane permeability of a pharmacological substance by introducing the polycationic derivative into the pharmacological substance via a chemical bond, but also a pharmacological substance whose biomembrane permeability is improved by the introduction of the polycationic derivative.

Claims

1. A compound having a structure represented by general formula (1) or a salt thereof, General formula (1) Wherein, n is an integer from 2 to 100; P represents a (C1-C5) alkylene group substituted with an amino group; Q is a structure represented by the following general formula (2) or the following general formula (3), In the above general formula (2), X represents an oxygen atom, an imino group, a sulfur atom, NHCO or CONH, and Y 1 and Y 2 may be the same or different, and represent an alkylene group having 1 to 6 carbon atoms, an oxygen atom or an imino group which may be substituted by a substituent selected from a halogen atom, a hydroxyl group, an amino group, a mercapto group, a carboxyl group and a sulfo group, and m is an integer from 1 to 30; In the above general formula (3), X represents an oxygen atom, an imino group, a sulfur atom, NHCO or CONH, Y represents an alkylene group having 1 to 6 carbon atoms, an oxygen atom or an imino group which may be substituted with a substituent selected from a halogen atom, a hydroxyl group, an amino group, a mercapto group, a carboxyl group and a sulfo group, and m is an integer from 1 to 30; Z represents a click functional group, and the click functional group is an azido group, an alkenyl group, an alkynyl group, a nitrile group, a thiol group, a maleimide group, an epoxy group, an aziridine group or a thioethylene group, However, it does not include:

2. The compound or salt thereof according to claim 1, which is represented by the general formula (4): Wherein, n is an integer from 2 to 100; m is an integer from 1 to 30; X is an oxygen atom or an imino group; Y represents an alkylene group having 1 to 6 carbon atoms which may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, an amino group, a mercapto group, a carboxyl group and a sulfo group; Z represents a click functional group.

3. A method for producing the compound or a salt thereof according to claim 1, comprising: A step of culturing a bacterium producing ε-poly-L-lysine, ε-poly-D-lysine, ε-poly-L-β-lysine, ε-poly-D-β-lysine, δ-poly-L-ornithine, δ-poly-D-ornithine, δ-poly-L-β-ornithine, δ-poly-D-β-ornithine, γ-poly-L-diaminobutyric acid, γ-poly-D-diaminobutyric acid, β-poly-L-diaminopropionic acid or β-poly-D-diaminopropionic acid in the presence of a compound represented by the general formula (2a) or the general formula (3a) or a salt thereof, In the above general formula (2a), X, Y 1 , Y 2 , Z and m have the same meanings as in the above general formula (1), In the above general formula (3a), X, Y, Z and m have the same meanings as in the above general formula (1); and A step of collecting the produced compound according to claim 1 or a salt thereof.

4. A method for producing the compound or a salt thereof according to claim 2, comprising: A step of culturing ε-PL producing bacteria in the presence of a compound represented by the general formula (3a) or a salt thereof, In the above general formula (3a), X represents an oxygen atom or an imino group, Y represents an alkylene group having 1 to 6 carbon atoms which may be substituted by a substituent selected from a halogen atom, a hydroxyl group, an amino group, a thiol group, a carboxyl group and a sulfo group, Z represents a click functional group, and m represents an integer from 1 to 30; and A step of collecting the produced compound according to claim 2 or a salt thereof.

5. A pharmacological substance, wherein: The compound according to claim 1 or a pharmaceutically acceptable salt thereof is introduced via a chemical bond.

6. A pharmacological substance, wherein: A compound represented by the following general formula (4) or a pharmaceutically acceptable salt thereof is introduced through a chemical bond, Wherein, n is an integer from 2 to 100, m is an integer from 1 to 30, X represents an oxygen atom or an imino group, Y represents an alkylene group having 1 to 6 carbon atoms which may be substituted with a substituent selected from a halogen atom, a hydroxyl group, an amino group, a mercapto group, a carboxyl group and a sulfo group, Z represents a click functional group.

7. The substance according to claim 5, wherein The pharmacologically active substance is at least one of an antibacterial agent, an antimicrobial antibiotic agent, and an anticancer agent.

8. The substance according to claim 7, wherein The antibacterial agent is (A) an antifungal agent; the antibacterial and antibiotic agent is (B) a β-lactam antibiotic, (C) an aminoglycoside antibiotic, (D) a tetracycline antibiotic, or (E) a peptide antibiotic, or (F) an antibacterial and antibiotic agent capable of introducing a click functional group organically or biochemically; or the anticancer agent is (G) an anticancer agent capable of introducing a click functional group organically or biochemically.

9. The substance according to claim 8, wherein (A) the antifungal agent is (a-1) a polyene antibiotic selected from amphotericin B, nystatin, chloramphenicol and pimacin, (a-2) micafungin, (a-3) itraconazole, (a-4) ketoconazole, (a-5) fluconazole, (a-6) miconazole, or (a-7) flucytosine; (B) the β-lactam antibiotic is (b-1) penicillin, (b-2) ampicillin, (b-3) talancillin, (b-4) bacillin, (b-5) cephalosporin C, (b-6) cephalexin, (b-7) cephradine, (b-8) cephamycin, or (b-9) imipenem; (C) the aminoglycoside antibiotic is (c-1) gentamicin, (c-2) kanamycin, (c-3) tobramycin, (c-4) amikacin, (c-5) arbekacin, or (c-6) nucleomycin; (D) the tetracycline antibiotic is (d-1) oxytetracycline, or (d-2) tetracycline, (E) the peptide antibiotic is (e-1) bacitracin, (e-2) gramicidin, (e-3) polymyxin B, (e-4) vancomycin, or (e-5) teicoplanin; (F) an antimicrobial antibiotic having an amino group, an imino group, a carboxyl group, a hydroxyl group, a carbonyl group, a thiol group, a phosphate group, an aldehyde group, etc., as an antimicrobial antibiotic into which a click functional group can be introduced organically or biochemically; or (G) As an anticancer agent capable of introducing a click functional group organically or biochemically, it is an anticancer agent having any one of an amino group, an imino group, a carboxyl group, a hydroxyl group, a carbonyl group, a thiol group, a phosphate group, an aldehyde group, etc., for example, (f-1) actinomycin D, (f-2) mitomycin, (f-3) an anthracycline antitumor drug selected from doxorubicin, daunorubicin, and aclarubicin, (f-4) bleomycin, or (f-5) cisplatin.

10. The substance according to claim 5 or 6, wherein The pharmacological agent is at least one of a protein and a gene.

11. The substance according to claim 10, wherein Proteins and genes can be organically or biochemically introduced with click functional groups.

12. The method for producing a pharmacologically active substance according to claim 6, characterized in that: The compound represented by the general formula (4) according to claim 6 or a pharmaceutically acceptable salt thereof is introduced into a pharmacologically active substance through a chemical bond.

13. The substance according to any one of claims 6 to 9, which is amphotericin B modified with ε-PL by click chemistry, as represented by general formula (5), Wherein n is an integer from 2 to 100.

14. The substance according to any one of claims 6 to 9, which is doxorubicin modified with ε-PL by click chemistry and represented by the general formula (5-2), Wherein n is an integer from 2 to 100.

15. The substance according to any one of claims 6 to 9, which is a monomeric Azami-Green fluorescent protein modified by click chemistry ε-PL represented by general formula (5-3), Wherein, n is an integer from 2 to 100, formula (5-3)' The group shown is of formula (5-3)" The monomeric Azami-Green fluorescent protein shown has one hydrogen atom removed.

16. Use of a compound represented by the general formula (2a) or (3a) or a salt thereof for producing the compound represented by the general formula (1) or a salt thereof according to claim 1.

17. Use of a compound represented by general formula (3a) or a salt thereof for preparing a compound represented by general formula (4) or a salt thereof according to claim 6, X represents an oxygen atom or an imino group, Y represents an alkylene group having 1 to 6 carbon atoms, Z represents a click functional group, and m represents an integer of 1 to 30.

18. A method for improving the biomembrane permeability of a pharmacological substance, characterized in that: The compound represented by the general formula (1) according to claim 1 or a salt thereof is introduced into a pharmacologically active substance.

19. Use of the compound represented by the general formula (1) or a salt thereof according to claim 1 for producing a pharmacological substance that improves biomembrane permeability.

20. A method for improving the water solubility of a pharmacological substance, characterized in that: The compound represented by the general formula (1) according to claim 1 or a salt thereof is introduced into a pharmacologically active substance.

21. Use of the compound represented by the general formula (1) or a salt thereof according to claim 1 for producing a pharmacological substance having improved water solubility.

22. A method for imparting at least one effect selected from the group consisting of antimicrobial activity, cultured cell adhesion effect, drip prevention effect, and antistatic function to an industrial substrate, characterized in that: The compound represented by the general formula (1) according to claim 1 or a salt thereof is introduced into an industrial substrate.

23. Use of the compound represented by the general formula (1) according to claim 1 or a salt thereof for manufacturing an industrial substrate imparted with at least one effect selected from the group consisting of antibacterial activity, cultured cell adhesion effect, anti-drip effect and antistatic function.

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