JP6495714B2 - Novel membrane permeable peptide - Google Patents

Description

  The present invention relates to novel membrane permeable peptides. More specifically, the present invention relates to a membrane-permeable peptide that functions as a drug delivery carrier composed of a non-natural amino acid having a cationic functional group in a side chain.

There is a great deal of research on techniques for introducing proteins, genes, etc. into cells. In these studies, delivery systems using macromolecules and peptides, and those using physical stimulation such as ultrasound and electricity have been developed. Although some reagents have been put to practical use, those utilizing oligopeptides found in proteins, or those utilizing natural amino acids with reference to known peptide sequences are predominant.
As typified by polyethylenimine, polycations having an ethylenediamine structure are well known to be efficient gene delivery carriers (Non-Patent Document 1), and further enhance the high transfection efficiency of these carriers. A number of studies have been conducted to raise and reduce cytotoxicity (Non-patent Document 2). A detailed mechanism involved in efficient transfection with polycations having an ethylenediamine structure has recently been reported (Non-patent Document 3). The degree of protonation plays a crucial role in high endosomal escape ability. The membrane destabilizing ability of monoprotonated gauche-type structures at neutral pH is low. On the other hand, the membrane destabilizing ability of the diprotonated anti-type structure at acidic pH is high and causes high endosomal escape with almost no cytotoxicity. A diprotonated ethylenediamine structure with high cationic charge density has the ability to interact with the cell membrane and deliver the cargo to the cell. Furthermore, peptides rich in arginine (Arg) have been identified as one of the most efficient cell membrane permeable peptides (CPPs) in the delivery of drugs, proteins, nucleic acids and nano-sized substances (Non-patent Documents 4 and 5) ). The cationic guanidino group in the Arg side chain is important for cell permeability. Therefore, new CPPs have been developed based on Arg-rich peptides and their derivatives (Non-patent Documents 6 and 7).
However, development of a delivery system that can satisfy all with low toxicity and high efficiency (low concentration) has not yet been achieved.

O. Bussif, F. Lezoalc'h, M. A. Zanta, M. D. Mergny, D. Scherman, B. Demeneix, J. P. Behr, Proc. Natl. Acad. Sci. USA 1995, 92, 7297-7301 Y. Lee, K. Kataoka, Adv. Polym. Sci. 2012, 249, 95-134 K. Miyata, M. Oba, M. Nakanishi, S. Fukushima, Y. Yamasaki, H. Koyama, N. Nishiyama, K. Kataoka, J. Am. Chem. Soc. 2008, 130, 16287-16294 P. A. Wender, D. J. Mitchell, K. Pattabiraman, E. T. Pelkey, L. Steinman, J. B. Rothbard, Proc. Natl. Acad. Sci. USA 2000, 97, 13003-13008 S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y. Sugiura, J. Biol. Chem. 2001, 276, 5836-5840 N. Umezawa, M. A. Gelman, M. C. Haigis, R. T. Raines, S. H. Gellman, J. Am. Chem. Soc. 2001, 124, 368-369 Y. A. Fillon, J. P. Anderson, J. Chmielewski, J. Am. Chem. Soc. 2005, 127, 11798-11803

  An object of the present invention is to provide novel cell membrane-permeable peptides and non-naturally-occurring amino acids that constitute them, which can function as drug delivery carriers.

In view of the above problems, the present inventors have designed and synthesized various non-natural amino acids suitable for synthesizing a cell membrane permeable peptide having high cell permeability even at physiological pH. As a result, by utilizing an amino acid having a guanidinylalkylamino group as a cationic functional group in the side chain, a peptide exceeding the function of the existing membrane permeable peptide is successfully developed to complete the present invention. It came to
The present invention is as follows.
[1] Formula (I):

Wherein, _{n 1} represents an integer of 1 to 3; _{n 2} represents an integer of ^1~6; R 1 ~R ³ is the same or different, a protecting group of a hydrogen atom or an amino group; ^{R 4} Is a hydrogen atom, a protecting group of an amino group, an amino acid residue or an acetyl group; R ⁵ is an amino acid residue, -NR ₂ , -OR or -SR (wherein R represents a hydrogen atom or a lower alkyl group) Is]
A compound represented by
[2] The compound of the above-mentioned [1], wherein R ^{1 to} R ³ are the same or different and are a protecting group of an amino group.
[3] The compound of the above-mentioned [1], wherein R ^{1 to} R ³ are hydrogen atoms.
[4] An oligopeptide consisting of 5 to 15 amino acid residues, wherein at least 20% or more of all the amino acid residues of the peptide are derived from the compound according to any one of the above [1] to [3] An oligopeptide that is
[5] At least 20% or more of all amino acid residues have an amino acid residue of the formula (I '):

(In the formula, the definition of each symbol is the same as the above [1])
The oligopeptide of the above-mentioned [4], which is derived from the compound represented by
[6] Formula (II):
(Xaa) n (II)
[Wherein, n is any integer of 5 to 15; n Xaa's are the same or different and any amino acid residue]
An oligopeptide represented by
At least one in the case of n = 5, at least two in the case of n = 6 to 10, and at least three Xaa in the case of n = 11 to 15:

(Wherein, n ₁ represents an integer of ₁ to 3; n ₂ represents an integer of 1 to 6; R ^{1 to} R ³ are the same or different and are a hydrogen atom or a protecting group of an amino group; * represents Show binding site)
An oligopeptide having a partial structure represented by
[7] A compound formed by combining the oligopeptide according to any one of the above [4] to [6] with a molecule X (X represents a physiologically active substance or a labeling compound).
[8] A composition comprising the oligopeptide according to any one of the above [4] to [6] and a molecule X (X represents a physiologically active substance or a labeling compound).
[9] The compound of the above-mentioned [7], wherein X is a physiologically active substance.
[10] The composition of the above-mentioned [8], wherein X is a physiologically active substance.
[11] A pharmaceutical composition comprising the compound of the above-mentioned [9] and / or the composition of the above-mentioned [10].
[12] The compound of the above-mentioned [7], wherein X is a labeling compound.
[13] The composition of the above-mentioned [8], wherein X is a labeling compound.
[14] A reagent for cell labeling, which comprises the compound of the above-mentioned [12] and / or the composition of the above-mentioned [10].
[15] A cell membrane permeable peptide having a guanidinylalkylamine (alkyl is 1 to 3 carbon atoms) structure in a side chain.

  The peptide synthesized from the non-natural amino acid of the present invention having a guanidinylalkylamino group (alkyl is 1 to 3 carbon atoms) which is a cationic functional group in the side chain is more effective than the conventional membrane permeable peptide. It can exhibit its function as a drug delivery carrier at low concentration. Therefore, it can be used as a drug delivery carrier without side effects such as toxicity. The present invention makes it possible to develop reagents that introduce proteins such as antibodies and genes such as plasmid DNA and siRNA into cells and safer and more effective pharmaceuticals.

FIG. 2 shows the structures of peptides 1 to 4 and compounds 19 and 22. It is the figure which showed the MALDI-TOF-MS result and HPLC chart of peptide 1. It is the figure which showed the MALDI-TOF-MS result and HPLC chart of peptide 2. It is the figure which showed the MALDI-TOF-MS result and HPLC chart of peptide 3. It is the figure which showed the MALDI-TOF-MS result and HPLC chart of peptide 4. It is the graph which showed the alpha / pH curve of compound 19 * 2 HCl ((circle)) and compound 22 ((circle)) obtained by potentiometric titration. Intracellular uptake (a, c, e) of peptides 1 to 4 using Huh-7 cells (a, b), HeLa cells (c, d) and CHO-K1 cells (e, f) and cell survival It is a graph which shows the result of having investigated rate (b, d, f). The peptide concentration dependency of the state after 2 hours of incubation was examined. Error bars indicate standard deviations, n = 3 (a, c, e) and n = 5 (b, d, f). It is a graph showing the result of having investigated the cellular uptake of peptides 1-4 to Huh-7 cells (a) and HeLa cells (b). The incubation time dependence in the state of a peptide concentration of 1 μM was examined. Error bars indicate standard deviation, n = 3. It is a graph showing the result of having investigated the effect (peptide concentration: 1 μM) of various endocytosis inhibitors in internalization of peptide 2 (a) and peptide 4 (b). Error bars indicate standard deviation, n = 3. * P <0.05 and ** P <0.01. It is a graph which shows the result of having investigated about intracellular co-localization with peptide 5 and peptide 2 or 4. Peptide 2 or 4 colocalized with peptide 5 was quantified. The results for Huh-7 cells are shown in (a), the results for HeLa cells are (b), and the results for CHO-K1 cells are (c). Error bars indicate the standard deviation, n = 20. *** P <0.001. It is a graph which shows the result of having investigated the intracellular distribution of peptide 2 and 4. Peptide 2 or 4 co-localized with Lysotracker green was quantified. Error bars indicate standard deviation, n = 18. It is a graph which shows the result of having investigated the gene transfer efficiency of the peptides 1-4. As a transgene, pDNA encoding a luciferase protein was used. Two types of Huh-7 cells and CHO-K1 cells were used, and TurboFect, a commercially available gene transfer reagent, was used as a positive control. The upper part shows the result of gene transfer efficiency and the lower part shows the result of cytotoxicity. The concentration of the peptide to pDNA was evaluated at 1-fold (1), 2-fold (2), 4-fold (4) and 8-fold (8). A single administration of pDNA was used as a background. Peptide 1 (Lys), Peptide 2 (Arg), Peptide 3 (Lys (Aet)), Peptide 4 (Lys (GEt)).

Hereinafter, the present invention will be described more specifically.
The present invention provides a non-naturally occurring amino acid having a guanidinylalkylamino group (alkyl having 1 to 3 carbon atoms) as a cationic functional group in the side chain (hereinafter, also referred to as the non-naturally occurring amino acid of the present invention) . The non-naturally occurring amino acid is specifically represented by the following formula (I) (hereinafter, also referred to as non-naturally occurring amino acid (I) of the present invention).

Wherein, _{n 1} represents an integer of 1 to 3; _{n 2} represents an integer of ^1~6; R 1 ~R ³ is the same or different, a protecting group of a hydrogen atom or an amino group; ^{R 4} Is a hydrogen atom, a protecting group of an amino group, an amino acid residue or an acetyl group; R ⁵ is an amino acid residue, -NR ₂ , -OR or -SR (wherein R represents a hydrogen atom or a lower alkyl group) Is]

The "amino group protecting group" is not particularly limited, and 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), 3-nitro-2-pyridinesulfenyl (Npys), benzyl Oxycarbonyl (Cbz), dansyl (DNS), isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, benzyl chloroformate (Cl-Cbz), benzyl bromoformate (Br-Cbz), adamantyloxycarbonyl, trifluoroacetyl, Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothiol, etc. are used. The protecting group of the amino group in R ^{1 to} R ⁴ may be the same or different, but when R ^{1 to} R ⁴ is a protecting group of amino group, R ^{1 to} R ³ is preferably Boc. , R ⁴ is preferably Fmoc.

  Examples of the "lower alkyl group" include linear or branched alkyl groups having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, 1- Examples include methylpropyl, pentyl, isopentyl, 1,2-dimethylpropyl, hexyl, 2-methylpentyl, 3-methylpentyl, 1,2-dimethylbutyl and the like, with preference given to methyl.

  The “amino acid residue” may be either α-amino acid or any other amino acid. As an α-amino acid residue, L-arginine, L-alanine, L-asparagine, L-aspartate, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine Amino acids such as L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, or L-cysteine or optical isomers thereof (D-form) The other amino acid residues are ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, γ-aminobutyric acid, ε-aminohexanoic acid, 6-aminohexanoic acid 2-aminoisobutyric acid, 3-aminopropionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, τ-butylglycine, τ-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acid, β-methyl amino acid And amino acid residues such as C-methyl amino acid and N-methyl amino acid, but all are not limited thereto.

As the non-natural amino acid (I) of the present invention, preferably, R ^{1 to} R ³ are protecting groups of amino group (eg, Boc), R ⁴ is a protecting group of amino group (eg, Fmoc), R ^{The following} compounds wherein ⁵ is -OR (e.g. -OH).

(Wherein the definition of each symbol is as described above)

As the non-naturally occurring amino acid (I) of the present invention, the following compounds in which all of R ^{1 to} R ⁴ are hydrogen atoms and R ⁵ is —OR (R is a hydrogen atom) are also preferable.

(Wherein the definition of each symbol is as described above)

The present invention provides an oligopeptide having a nonnatural amino acid having a guanidinylalkylamino group (alkyl is 1 to 3 carbon atoms) as a cationic functional group in the side chain (hereinafter referred to as "oligopeptide according to the present invention") Called). Examples of non-naturally occurring amino acids having a guanidinylalkylamino group (alkyl is 1 to 3 carbon atoms) include the non-naturally occurring amino acid (I) of the present invention.
The oligopeptide preferably consists of 5 to 15, more preferably 6 to 14, more preferably 7 to 13, particularly preferably 8 to 12 amino acid residues, and at least 20% or more of the total amino acid residues of the peptide. Is a non-naturally occurring amino acid having a guanidinylalkylamino group (alkyl is 1 to 3 carbon atoms). An example is an oligopeptide in which at least 20% or more of the amino acid residues of all the amino acid residues are derived from the non-naturally occurring amino acid represented by formula (I).

As one embodiment of the oligopeptide of the present invention, the formula (II):
(Xaa) n (II)
[Wherein, n is any integer of 5 to 15; n Xaa's are the same or different and any amino acid residue]
The compound represented by these is mentioned. The compound has the following partial structure derived from the non-naturally occurring amino acid (I) of the present invention, at least one when n = 5, at least two when n = 6 to 10, and at least There are three.

(Wherein, n ₁ represents an integer of ₁ to 3; n ₂ represents an integer of 1 to 6; * represents a binding site)
That is, the oligopeptide represented by the formula (II) is a non-naturally occurring amino acid (preferably, the non-naturally occurring form of the present invention) having a guanidinylalkylamino group (alkyl is 1 to 3 carbon atoms) in its amino acid sequence. The amino acid (I) has at least 20% or more of all amino acid residues, and when there are a plurality of such non-naturally occurring amino acids, they are contained in the amino acid sequence either continuously or discontinuously. Amino acid residues other than non-natural amino acids (preferably, non-natural amino acid (I) of the present invention) having a guanidinyl alkylamino group (alkyl is 1 to 3 carbon atoms) are α-amino acids and other amino acids And any of the various amino acid residues described above.
Both ends of the oligopeptide represented by formula (II) may be optionally modified, for example, it may be protected with an amino protecting group or may be acetylated.

The terminal or side chain of the peptide of the present invention may be linked to the molecule X depending on the purpose of use of the peptide. Also, molecule X can be linked to the peptide as nanoparticles. The molecule X is not particularly limited as long as it is a substance that the cellular uptake of the peptide of the present invention is desired to take into cells, and includes physiologically active substances and labeling compounds. When X is a physiologically active substance, the peptide of the present invention can be a pharmaceutical composition, and when X is a compound for labeling, the peptide of the present invention can be a reagent for cell labeling.
The “physiologically active substance” may be, for example, a substance capable of affecting the function or state of the cell when introduced into the cell, such as a nucleic acid, a peptide, a protein, a lipid, a peptide lipid, Examples include sugars, low molecular weight compounds, and other synthetic or natural compounds. More specifically, enzymes, antibodies or fragments thereof, glycoproteins, etc. may also be used as molecule X. When delivery into cells is for the treatment and / or prevention of a disease, the substance has a treatment and / or prophylaxis activity for the disease, for example, an antihypertensive agent, an antihypertensive agent, an antipsychotic Agents, analgesics, antidepressants, antidepressants, anti-anxiety agents, sedatives, hypnotics, antidepressants, opioid agonists, anti-asthma agents, anesthetics, anti-arrhythmic agents, anti-arthritis agents, anticonvulsants, ACE inhibitors, congestion Removal agents, antibiotics, antianginal agents, diuretics, antiparkinsonian agents, bronchodilators, labor promoting agents, antidiuretics, antihyperlipidemic agents, immunosuppressants, immunomodulators, antiemetics, Anti-infective agent, anti-neoplastic agent, anti-fungal agent, antiviral agent, anti-diabetic agent, anti-allergic agent, antipyretic agent, antineoplastic agent, anti-gout agent, antihistamine agent, antipruritic agent, bone regulator, cardiovascular agent, Cholesterol-lowering agents, antimalarials, quit smoking Drugs, antitussives, expectorants, mucolytics, drugs for nasal congestion, dopamine agonists, drugs for gastrointestinal tract, muscle relaxants, neuromuscular blockers, parasympathomimetics, prostaglandins, stimulants, appetite suppressants And thyroid or antithyroid agents, hormones, anti-migraine agents, anti-obesity agents, anti-inflammatory agents and the like.

  In a preferred embodiment, the physiologically active substance that can be introduced into cells is a nucleic acid. The nucleic acid is not particularly limited, and may be any of DNA, RNA, chimeric nucleic acid of DNA and RNA, hybrid of DNA / RNA, and the like. The nucleic acid may be any one of 1 to 3 strands, but preferably 1 or 2 strands. The nucleic acid is another type of nucleotide that is an N-glycoside of a purine or pyrimidine base, or another oligomer having a non-nucleotide backbone (eg, a commercially available peptide nucleic acid (PNA), etc.) or other oligomer containing special linkages (However, the oligomer may contain a nucleotide having an arrangement that permits base pairing or attachment of a base as found in DNA or RNA). Furthermore, additions of known modifications, such as those known in the art with a label, capped, methylated, substituted with one or more naturally occurring nucleotides by analogues, Intramolecular nucleotide-modified ones, for example, those having non-charged bonds (eg, methyl phosphonate, phosphotriester, phosphoramidate, carbamate etc.), charged bonds or sulfur-containing bonds (eg, phosphorothioate, phosphoro) Those having a dithioate, such as those having a side chain group such as proteins (nuclease, nuclease inhibitors, toxins, antibodies, signal peptides, etc.) or sugars (eg, monosaccharides, etc.), Intercurrent compounds ( Eg with acridine, psoralen etc), chelation Containing a metal (eg, metal, radioactive metal, boron, oxidizing metal, etc.), containing an alkylating agent, having a modified bond (eg, nucleic acid of an α-anomeric type, etc.) It may be

  For example, the type of DNA can be appropriately selected according to the purpose of use and is not particularly limited, and examples include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC and the like, preferably plasmid DNA , CDNA, antisense DNA. Circular DNA such as plasmid DNA is appropriately digested with a restriction enzyme or the like and can also be used as linear DNA. Further, the type of RNA can be appropriately selected according to the purpose of use, and is not particularly limited. For example, siRNA, miRNA, shRNA, antisense RNA, messenger RNA, single stranded RNA genome, double stranded RNA genome And RNA replicon, transfer RNA, ribosomal RNA and the like, preferably siRNA, miRNA, shRNA, mRNA, antisense RNA, RNA replicon.

Examples of the “labeling compound” include, but are not limited to, various enzymes, fluorescent substances, luminescent substances, bioluminescent substances and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase. As examples of suitable fluorescent substances, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, tetrarhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin can be mentioned. Luminol can be mentioned as an example of the luminescent material. As examples of bioluminescent substances, mention may be made of luciferase, luciferin and aequorin. As examples of suitable radioactive substances, ¹²⁵ I, ¹³¹ I, ³⁵ S and ³ H can be mentioned.

  The uptake of molecule X into cells is carried out by simply mixing molecule X with the oligopeptide of the present invention other than the method of linking molecule X to the oligopeptide of the present invention and providing the resulting compound to cells as described above. It can also be carried out by the method of providing it to cells.

  The molecule X may be linked or mixed with the oligopeptide of the present invention as it is, but as a nanoparticle, it may be linked or mixed with the oligopeptide of the present invention.

  The non-naturally occurring amino acid (I) of the present invention can be produced, for example, by the method shown in the following scheme. The protection of functional groups that should not participate in the reaction of the raw materials, protection of the functional groups, and removal of the protective groups, activation of functional groups involved in the reaction, etc. may be appropriately selected from known groups or known means. In the following scheme, specific raw materials, specific reagents and reaction conditions are described as an example, but it is possible to substitute other similar raw materials and reagents, and the reaction conditions are also appropriately set depending on the raw materials and reagents used. It will be apparent to those skilled in the art.

  The oligopeptide of the present invention may be synthesized by, for example, either solid phase synthesis or liquid phase synthesis. That is, the target oligopeptide can be produced by condensing the partial peptide or amino acid capable of constructing the oligopeptide of the present invention and the remaining portion, and removing the protecting group if the product has a protecting group. .

  More specifically, a commercially available resin for peptide synthesis can be used for the synthesis of the oligopeptide of the present invention. As such resin, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylphenyl Acetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2', 4'-dimethoxyphenyl-Fmoc aminoethyl) phenoxy resin and the like can be mentioned.

  For condensation of the protected amino acids described above, various activation reagents that can be used for peptide synthesis can be used, and uroniums are particularly preferable. As uroniums, COMU, HATU, HBTU etc. are used. For activation with these, protected amino acids are added directly to the resin together with racemization suppressing additives (eg HOBt, HOOBt), or the protected amino acids are activated beforehand as symmetrical acid anhydrides or HOBt esters or HOOBt esters. Can then be added to the resin.

  The solvent used for the activation of the protected amino acid and the condensation with the resin may be appropriately selected from the solvents known to be usable for the peptide condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide and the like Sulfoxides, ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or suitable mixtures thereof are used.

  The reaction temperature is appropriately selected from the range known to be applicable to peptide bond formation reactions, and is usually selected appropriately from the range of about -20 ° C to 100 ° C. The activated amino acid derivative is usually used in 1.5 to 4-fold excess. As a result of the test using ninhydrin reaction, when condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protective group. Acetic anhydride or acetylimidazole can be used to acetylate unreacted amino acids when repeated reactions do not result in sufficient condensation.

  As a protecting group of the amino group of the raw material, for example, Z, Boc, tert-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothiol, Fmoc and the like are used.

  The carboxyl group is, for example, an alkyl esterified (eg, methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl and the like, linear, branched or cyclic alkyl esters) Aralkylation (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacylation, benzyloxycarbonyl hydrazide, tert-butoxy It can be protected by carbonyl hydrazide, trityl hydrazide and the like.

  The hydroxyl group possessed by serine, threonine and the like can be protected by, for example, esterification or etherification. As a group suitable for this esterification, for example, a lower alkanoyl group such as acetyl, an aroyl group such as benzoyl, a group derived from carbonic acid such as benzyloxycarbonyl, ethoxycarbonyl and the like are used. Moreover, as a group suitable for etherification, it is benzyl, tetrahydropyranyl, tert- butyl etc., for example.

As a protective group of the phenolic hydroxyl group which tyrosine etc. have, Bzl, Cl2-Bzl, ₂ -nitrobenzyl, Br-Z, tert- butyl etc. are used, for example.

  As a protective group of imidazole possessed by histidine etc., for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc etc. are used.

  As a protective group of indole which tryptophan and the like have, for example, formyl, 2,4,6-trimethylbenzenesulfonyl, 2,4,6-trimethoxybenzenesulfonyl, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, β, β, β-trichloroethyloxycarbonyl, diphenylphosphinothioyl t-butoxycarbonyl and the like are used.

  The protective group for the thiol group possessed by cysteine etc. is, for example, paramethoxybenzyl, 4-methylbenzyl, benzyl, t-butyl, adamantyl, trityl, acetamidomethyl, carbomethoxysulfenyl, 3-nitro-2-pyridinesulfenyl Etc. are used.

  As a guanidino group protecting group which arginine etc. have, for example, nitro, tosyl, p-methoxybenzene sulfonyl, mesitylene sulfonyl, pentamethyl benzene sulfonyl, 4-methoxy-2,3,6-trimethyl benzene sulfonyl, carbobenzoxy, Isobornyloxycarbonyl, adamantyloxycarbonyl and the like are used.

  As a protecting group of the mercapto group which a methionine residue etc. have, substituted benzyl groups, such as benzyl and p-methoxy benzyl; Trityl; benzhydryl; acetamidomethyl; carbomethoxysulfenyl etc. are used.

  As activated ones of the carboxyl group of the raw material, for example, corresponding acid anhydrides, azides, active esters [alcohols such as pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, Examples thereof include cyanomethyl alcohol, paranitrophenol, HONB, esters with N-hydroxysuccinimide, HOBt) and the like. As the activated amino group of the raw material, for example, the corresponding phosphoric acid amide is used.

  As a method of removing (eliminating) a protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethane or the like Acid treatment with sulfonic acid, trifluoroacetic acid or a mixture thereof or the like, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine or the like, reduction with sodium in liquid ammonia, or the like may also be used. The elimination reaction by the acid treatment is generally carried out at a temperature of about -20 ° C to 40 ° C, but in the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 Addition of a cation scavenger such as butanedithiol, 1,2-ethanedithiol etc. is effective. In addition, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butanedithiol It is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like, in addition to deprotection by acid treatment in the presence of H 2 O, etc.

  The protection of functional groups that should not participate in the reaction of the raw materials, protection of the functional groups, and removal of the protective groups, activation of functional groups involved in the reaction, etc. may be appropriately selected from known groups or known means.

  After the reaction, the desired oligopeptide can be isolated and purified by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the peptide obtained by the above method is a free form, it can be converted to a suitable salt by a known method, and conversely, when it is obtained as a salt, it can be converted to a free form by a known method it can.

  As salts, physiologically acceptable acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) And salts with tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used. In addition, salts with inorganic bases (eg, alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, aluminum or ammonium etc.), organic bases (eg, trimethylamine, triethylamine, pyridine, picoline, 2, 6 -A salt with lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N'-dibenzylethylenediamine etc., and the like can also be mentioned.

  The oligopeptide of the present invention thus obtained is excellent in cell membrane permeability and can function as a drug delivery carrier. Therefore, the oligopeptide of the present invention is optionally linked or mixed with molecule X (bioactive substance, labeling compound). By linking to or mixing with the oligopeptide of the present invention and providing it to cells, the molecule X is suitably taken up into cells by the cell membrane permeability of the oligopeptide.

  The oligopeptide of the present invention can be formulated alone or according to a conventional means with a pharmacologically acceptable carrier, and used as a pharmaceutical composition or reagent composition (eg, a reagent for cell labeling). When the peptide of the present invention is formulated as a reagent composition, the peptide may be used as it is or, for example, water or other physiologically acceptable liquid (eg, saline, phosphate buffered saline (eg, PBS), an aqueous solvent such as a medium used in ordinary cell culture (eg RPMI 1640, DMEM, HAMF-12, Eagle's medium etc.), an organic solvent such as ethanol, methanol, DMSO or a mixture of an aqueous solvent and an organic solvent Etc.) may be provided as a sterile solution or suspension. The reagent composition can optionally contain physiologically acceptable excipients, vehicles, preservatives, stabilizers, binders and the like known per se.

  When the oligopeptide of the present invention is formulated as a pharmaceutical composition, the peptide may be used as it is or as a pharmaceutically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binding agent Orally (eg, granules, powders, tablets, pills, pills, capsules, syrups, emulsions, suspensions, etc.) or not by mixing in the unit dose form required for generally accepted formulation practice with Oral agents (eg, injections (eg, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drops, external preparations (eg, sprays, patches, ointments), suppositories, etc. Can be manufactured as

Examples of additives that can be incorporated into tablets and capsules include, for example, gelatin, corn starch, tragacanth, binders such as gum arabic, excipients such as crystalline cellulose, and swelling such as corn starch and gelatin. Agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavors such as peppermint, red mono oil or cherry, etc. may be used. When the dosage unit form is a capsule, it may further contain a liquid carrier such as fat and oil in the above-mentioned type of material. As the aqueous solution for injection, for example, physiological saline, glucose, and isotonic solution containing other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.), etc. are used, and appropriate dissolution assistance is provided. It may be used in combination with an agent such as alcohol (eg ethanol), polyalcohol (eg propylene glycol, polyethylene glycol), nonionic surfactant (eg polysorbate ^80TM , HCO-50) and the like. As an oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with a solubilizer such as benzyl benzoate and benzyl alcohol.

  In addition, the above-mentioned pharmaceutical composition includes, for example, buffer (for example, phosphate buffer, sodium acetate buffer and the like), soothing agent (for example, benzalkonium chloride, procaine hydrochloride and the like), stabilizer (for example, human) It may be blended with serum albumin, polyethylene glycol etc., preservative (eg benzyl alcohol, phenol etc.), antioxidant (eg ascorbic acid etc) and the like.

  The administration method of the above-mentioned pharmaceutical composition to a subject is not particularly limited as long as the composition reaches and contacts the target cell and the physiologically active substance and the like contained in the composition can be introduced into cells. An administration method known per se (oral administration, parenteral administration (intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, intraperitoneal administration, etc.) in consideration of the type, type and site of target cells, etc. Spray etc.) can be suitably selected.

  The dosage of the above-mentioned pharmaceutical composition is not particularly limited as long as introduction of the physiologically active substance into cells can be achieved, and the kind of administration target, administration method, kind of physiologically active substance, type of target cell, Although it can be appropriately selected in consideration of the site etc., in the case of oral administration, in general, for example, in a human (as a 60 kg body weight), its single dose is about 0.001 mg to 10000 mg as a complex. When administered parenterally (e.g., intravenous administration, etc.), in general, for example, in a human (as 60 kg body weight), the single dose is about 0.0001 mg to 3000 mg as a complex.

  Targets of administration include vertebrates such as mammals including humans (humans, monkeys, mice, rats, hamsters, cattle, etc.), birds (chicken, ostrich, etc.), amphibians (frogs, etc.), fish (zebrafish, medakas, etc.) Preferably, it is a mammal, more preferably a human.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited by these examples. Unless otherwise indicated, reagents and the like used are commercially available.
The following measuring devices were used.
NMR: JEOL AL 400 (400 MHz)
Optical rotation: JASCO DIP-370, 0.5 dm cell using mass spectrometry: FAB-MS (JEOL JMS-700N); DART-MS (JEOL JMS-T1000TD)

Example 1: Synthesis of unnatural amino acid (I) of the present invention

[1] N-α-benzyloxycarbonyl-N-ε-tert-butoxycarbonyl-2'-aminoethyl-L-lysine methyl ester {Cbz-L-Lys [AEt (Boc)]-OMe, compound 8} synthesis Dess-Martin periodinane (DMP; 9.08 g, 21.4 mmol ) and 2- (tert- butoxycarbonylamino) -1- ethanol (compound 6) (2.88 g, 17.8 mmol ) CH 2 Cl 2 (100 mL) stirring It was added to the solution and the solution was stirred at room temperature for 2 hours. NaHCO ₃ -Na ₂ S in ₂ O ₃ by addition of a saturated aqueous solution of (100 mL), and stirred for 30 minutes at room temperature. The solution was extracted with CHCl ₃ , dried over MgSO _{4 and} evaporated in vacuo to give the crude aldehyde. Crude aldehyde, Cbz-L-Lys-OMe (5.25 g, 17.8 mmol) and and _CH 2 Cl ₂ (100 mL) solution of MS4Å (1 g) was stirred 1 hour at 0 ° C.. Sodium triacetoxyborohydride (4.80 g, 22.7 mmol) was added to the stirring solution and then stirred at room temperature overnight. The solution was extracted with CHCl ₃ and dried over MgSO ₄ . The solvent is removed to give a residue which is purified by column chromatography on silica gel (10% MeOH in CHCl ₃ ) as a colorless oil Cbz-L-Lys [AEt (Boc)]-OMe (Compound 8) (2.63 g, 34%) was obtained.
[α] _D ²⁵ = +2.92 (c 1.35, CHCl ₃ ); IR (neat) ₃ 3345, 2955, 1715, 1697, 1520, 1454, 1400, 1254, 1215, 1169, 1049 cm ^-1 ; ¹ H NMR ( 400 MHz, CDCl ₃ ) δ 7.29-7.36 (m, 5 H), 5. 75 (br s, 1 H), 5. 69 (br s, 1 H), 5. 10 (s, 2 H), 4. 35 (m, 1 H), 3.74 (s, 3H), 3.44 (br s, 1H), 3.31-3.35 (m, 2H), 2.84-2.90 (m, 2H), 2.74 (t, J = 7.10 Hz, 2H), 1.96-1.98 (m, 2H), 1.83 (m, 1 H), 1.60-1. 71 (m, 3 H), 1.43 (s, 9 H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 172.7, 156.5, 156.1, 136.2, 128.5, 128.13, 128.09, 80.0, 67.0, 53.6, 52.6, 48.5, 47.8, 37.3, 21.7, 25.5, 22.4; FAB (+) HRMS calcd for C ₂₂ H ₃₆ N ₃ O ₆ [M ⁺ + H]: 438.2604; found: 438.2596.

[2] N-α-benzyloxycarbonyl-N-ε- (tert-butoxycarbonyl-2'-aminoethyl) -N-ε-tert-butoxycarbonyl-L-lysine methyl ester {Cbz-L-Lys [Boc , AEt (Boc)]-OMe, Compound 9} Synthesis of Boc ₂ O (583 mg, 2.67 mmol) and Et ₃ N (270 mg, 2.67 mmol) with Compound 8 (973 mg, 2.22 mmol) in CH ₂ Cl ₂ (30 mL) was added to the stirred solution and the solution was stirred at room temperature for 5 hours. The solvent is removed to give a residue which is purified by column chromatography on silica gel (40% EtOAc in n-hexane) to give Cbz-L-Lys [Boc, AEt (Boc) as a colorless oil ] -OMe (compound 9) (820 mg, 69%) was obtained.
[α] _D ²⁵ = +1.99 (c 2.06, CHCl ₃ ); IR (neat) ₃ 3345, 2974, 1725, 1710, 1686, 1520, 1416, 1366, 1250, 1169, 1065 cm ^-1 ; ¹ H NMR ( 400 MHz, CDCl ₃ ) δ 7.28-7.36 (m, 5H), 5.37-5.52 (m, 1H), 5.10 (s, 2H), 4.80-5.04 (m, 1H), 4.35 (m, 1H), 3.74 (m) s, 3H), 3.17-3.24 (m, 6H), 1.84 (m, 1H), 1.69 (m, 1H), 1.50-1.53 (m, 2H), 1.44 (s, 9H), 1.43 (s, 9H) , 1.24-1.36 (m, 2H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 172.8, 156.0, 155.8, 136.2, 128.3, 79.7, 69.1, 66.8, 53.7, 52.2, 47.3, 46.3, 39.4, 32.2, 31.7 , 28.2, 27.4, 22.3; FAB (+) HRMS calcd for C ₂₇ H ₄₄ N ₃ O ₈ [M ⁺ + H]: 538.3128; found: 538.3132.

[3] Synthesis of N-α-benzyloxycarbonyl-N-ε-2'-aminoethyl-L-lysine methyl ester dihydrochloride {Cbz-L-Lys (AEt)}-OMe · 2HCl, compound 13} A solution of 9 (387 mg, 0.720 mmol) in 4 M HCl / dioxane (7.2 mL) was stirred at room temperature for 2 hours. The solvent was removed to obtain Cbz-L-Lys (AEt) -OMe · 2HCl (compound 13) (289 mg, 98%) as colorless crystals.
Mp 149-151 ° C .; [α] _D ²⁷ = -14.3 (c 1.11, MeOH); IR (KBr) 334 3341, 2978, 2936, 1720, 1524, 1450, 1420, 1366, 1250, 1165, 1076 cm ^{-1 1} H NMR (400 MHz, CD ₃ OD) δ 7.22-7.29 (m, 5H), 5.02 (s, 2H), 4.13 (m, 1H), 3.65 (s, 3H), 3.23-3.33 (m, 4H) ), 3.01 (t, J = 7.4 Hz, 2 H), 1.62-1. 85 (m, 4 H), 1.37-1. 50 (m, 2 H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 174.3, 158.6, 138.1, 129.5, 129.0, 128.8, 67.7, 55.2, 52.8, 49.6, 45.7, 36.9, 31.9, 26.7, 23.8; FAB (+) HRMS calcd for C ₁₇ H ₂₈ N ₃ O ₄ [M ⁺ + H]: 338.2080; found: 338.2032.

[4] N-α-benzyloxycarbonyl-N-ε- [N ', N''-bis (tert-butoxycarbonyl) -2'-guanidinyl] ethyl-L-lysine methyl ester {Cbz-L-Lys [ Synthesis of GEt (Boc) ₂ ] -OMe, compound 14} Compound 13 (289 mg, 0.681 mmol) was prepared by using 1,3-bis (tert-butoxycarbonyl) -2- (trifluoromethanesulfonyl) guanidine (320 mg, 0.817). It was added to a stirred solution of mmol) and Et ₃ N (83 mg, 0.817 mmol) in CH ₂ Cl ₂ (10 mL) and stirred overnight at room temperature. The solvent is removed to give a residue which is rapidly purified by short column chromatography on silica gel (8% MeOH in CHCl ₃ ) to give Cbz-L-Lys [GEt (Boc) ₂ as a colorless oil ] -OMe (compound 14) (362 mg, 89%) was obtained.
¹ H NMR (400 MHz, CHCl ₃ ) δ 11.42 (br s, 1 H), 8.76 (br s, 1 H), 7.29-7.36 (m, 5 H), 5.54 (br s, 1 H), 5.53 (d, J = 7.6 Hz, 1H), 5.11 (s, 2H), 4.35 (m, 1H), 3.74 (s, 3H), 3.53-3.60 (m, 2H), 2.98-3.04 (m, 2H), 2.75-2.79 (m , 2H), 1.85 (m, 1H), 1.66-1.75 (m, 3H), 1.50 (s, 9H), 1.48 (s, 9H), 1.21-1.58 (m, 2H).

[5] N-α-benzyloxycarbonyl-N-ε- [N ′, N ′ ′-bis- (tert-butoxycarbonyl) -2′-guanidinyl] ethyl-N-ε-tert-butoxycarbonyl-L- lysine methyl ester {Cbz-L-Lys [Boc , GEt (Boc) 2] -OMe, compound 15)} compound 14 (1.88 g _{of, 3.24 mmol), Boc 2 O} (849 mg, 3.89 mmol) and Et ₃ The mixture of N (394 mg, 3.89 mmol) was stirred at room temperature for 4 hours. The solvent is removed to give a residue which is purified by column chromatography on silica gel (40% EtOAc in n-hexane) to give Cbz-L-Lys [Boc, GEt (Boc) ₂ ]-as a colorless oil OMe (compound 15) (1.94 g, 88%) was obtained.
[α] _D ²¹ = +4.59 (c 1.14, CHCl ₃ ); IR (neat) ₃ 3333, 2978, 2936, 1728, 1690, 1682, 1616, 1574, 1535, 1415, 1366, 1335, 1254, 1231 ^{1 1} H NMR (400 MHz, CHCl ₃ ) δ 11.48 (br s, 1 H), 8.44 (m, 1 H), 7.31-7. 36 (m, 5 H), 5.36 -5.52 (m, 1H), 5.10 (s, 2H), 4.34 (m, 1H), 3.73 (s, 3H), 3.50-3.56 (m, 2H), 3.34-3.42 (m, 2H), 3.12-3.24 (m, 1 H), 1. 84 (m, 1 H), 1. 70 (m, 1 H), 1. 49 (s, 9 H), 1. 48 (s, 9 H), 1. 44 (s, 9 H), 1.27-1. 40 (m, 4 H); ¹³ C NMR (100 MHz, CHCl ₃ ) δ 172.8, 163.4, 156.3, 155.6, 153.0, 136.2, 128.4, 128.0, 83.0, 79.9, 79.1, 66.8, 53.7, 52.2, 46.0, 45.6, 39.4, 31.8, 28.23 , 28.18, 28.1, 27.9, 27.3, 22.2; FAB (+) HRMS calcd for C ₃₃ H ₅₄ N ₅ O ₁₀ [M ⁺ + H]: 680.3871; found: 680.3863.

[6] N-α- (9-fluorenylmethoxycarbonyl) -N-ε- [N ′, N ′ ′-bis- (tert-butoxycarbonyl) -2′-guanidinyl] ethyl-N-ε-tert Synthesis of -butoxycarbonyl-L-lysine methyl ester {Fmoc-L-Lys [Boc, GEt (Boc) ₂ ] -OH, compound 18} 0.1 M NaOH (30.4 mL, 3.04 mmol) aqueous solution to compound 15 (1.88 g , 2.76 mmol) in MeOH (30 mL) and the solution was stirred at room temperature for 24 h. After MeOH removal, acidify the solution to pH 2-3 with citric acid, extract with EtOAc, dry over Na ₂ SO ₄ , remove in vacuo and crude carboxylic acid (compound 16) (1.66 g, 90% Got). In MeOH (30 mL) in the compound 16 (1.66 g, 2.49 mmol) and a mixture of 5% Pd-C (500 mg ) was stirred vigorously at room temperature under an atmosphere of _{H 2.} After stirring overnight, the Pd-C catalyst was filtered off, and the filtrate was evaporated under reduced pressure to give crude amino acid (compound 17) (1.33 g, quantitative). A solution of Fmoc-OSu (925 mg, 2.74 mmol) in dioxane (20 mL) is added to a stirred solution of compound 17 (1.33 g, 2.49 mmol) and NaHCO ₃ (628 mg, 7.48 mmol) in water (20 mL), The solution was stirred at room temperature overnight. After dioxane removal, the solution was acidified with citric acid, extracted with EtOAc and dried over Na ₂ SO ₄ . The solvent was removed to give a white solid which was purified by chromatography on silica gel. The fraction eluted with 3% MeOH (in CHCl ₃ ) gave Fmoc-L-Lys [Boc, GEt (Boc) ₂ ] -OH (Compound 18) (972 mg, 52%) as colorless crystals.
Mp 93-94 ° C .; [α] _D ²⁰ = +12.1 (c 1.41, CHCl ₃ ); IR (KBr) ₃ 3333, 2979, 2936, 1721, 1686, 1639, 1620, 1420, 1366, 1331 cm ^-1 ; ¹ H NMR (400 MHz, CDCl ₃ ) δ 11.53 (br s, 1 H), 8. 49 (m, 1 H), 7. 76 (d, J = 7.2 Hz, 2 H), 7. 60 (d, J = 6.0 Hz, 2 H), 7.39 (t, J = 7.2 Hz, 2 H), 7.31 (t, J = 7.2 Hz, 2 H), 6.23 (br s, 1 H), 5.61 (m, 1 H), 4.35-4. 50 (m, 3 H), 4.22 ( t, J = 6.8 Hz, 1 H), 3.16-3.51 (m, 6 H), 1. 89-1. 97 (m, 2 H), 1. 48 (s, 9 H), 1. 47 (s, 9 H), 1. 45 (s, 9 H), 1. 20 -1.60 (m, 4H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 174.8, 163.1, 156.5, 156.0, 155.7, 153.9, 143.9, 141.3, 127.7, 127.0, 119.9, 83.3, 80.4, 79.6, 67.0 , 53.5, 47.1, 46.5, 46.0, 39.6, 31.1, 28.3, 28.2, 21.7; FAB (+) HRMS calcd for C ₃₉ H ₅₆ N ₅ O ₁₀ [M + + H]: 754.4027; found: 754.4050.

Example 2: Synthesis of peptide (II) of the present invention Various peptides were synthesized on a solid phase carrier by Fmoc solid phase method using standard commercially available Rink amide resin and Fmoc amino acid. The following describes a typical coupling and deprotection cycle on a 10 μmol scale.
First, 26.3 mg of CLEAR amide resin (loading 0.38 mmol / g) was soaked in DMF overnight. After DMF removal, 20% piperidine (in DMF) was added to the resin for deprotection. After removal of piperidine and washing off, Fmoc amino acid or 5 (6) -tetramethylrhodamine carboxylic acid (3 equivalents), COMU as coupling reagent (3 equivalents) and as a base, dissolved in DMF (1.0 mL) Coupling reaction was performed by adding DIPEA (6 equivalents). As Fmoc amino acids, Fmoc-Gly-OH and Fmoc-L-Lys [Boc, GEt (Boc) ₂ ] -OH (compound 18) synthesized in Example 1 were used. The resin was then suspended in a cleavage cocktail (TFA: 1.9 mL; H ₂ O: 50 μL; TIS: 50 μL). The TFA solution was evaporated to reduce the volume and added to cold diethyl ether to precipitate the peptide. The dried crude peptide was dissolved in acetonitrile and / or H ₂ O and then purified by RP-HPLC using COSMOSIL Packed Column 5C ₁₈ -AR-II (20 ID × 250 mm) (Nacalai). The red crystals are obtained by freeze-drying and the crystals are analyzed RP-HPLC (COSMOSIL Packed Column 5C ₁₈ -AR-II, 4.6 ID x 250 mm) and MALDI-TOF-MS (Bruker Daltonics Ultraflex, Fremont, CA) Identified by RP-HPLC utilized JASCO-PU-2089 Plus (JASCO) equipped with JASCO-2075-Plus as a detector. Solvent A: 0.05% TFA in H ₂ O, Solvent B: 0.05% TFA in Acetonitrile. Purification was performed under the conditions of flow rate 1.0 mL / min, detection at 220 nm, gradient (95% to 50% of solvent A, 20 minutes). The purity of the final compound was determined using the same RP-HPLC conditions (20 minutes with 95% to 35% solvent A, then 5 minutes with 35% to 10% solvent A) at a flow rate of 1.0 mL / min. It checked further.
The synthesized peptide was then purified using RP-HPLC. The homogeneity of the purified peptide was verified by analytical RP-HPLC and MALDI-TOF / MS. The results are shown in FIG. 2D. The resulting labeled peptide may be hereinafter also abbreviated as TMR-Gly- [L-Lys (GEt)] ₉ -NH ₂ (peptide 4).

Comparative Example 1: N-α- (9-fluorenylmethoxycarbonyl) -N-ε- (tert-butoxycarbonyl-2'-aminoethyl) -N-ε-tert-butoxycarbonyl-L-lysine {Fmoc- Synthesis of peptide starting from L-Lys [Boc, AEt (Boc)]-OH}

Cbz-L-Lys [Boc, AEt (Boc)]-OMe (compound 9) was synthesized in the same manner as in [1] and [2] of Example 1.
An aqueous solution of 0.1 M NaOH (27.1 mL, 2.71 mmol) was added to a stirred solution of compound 9 (1.22 g, 2.26 mmol) in MeOH (10 mL) and the solution was stirred at room temperature for 2 hours. After MeOH removal, acidify the solution to pH 2-3 with 1 M aqueous NaHSO ₄ solution, extract with EtOAc, dry over Na ₂ SO _{4 and} remove in vacuo to give crude carboxylic acid (Compound 10) (1.00) g, 84%). MeOH (30 mL) solution of compound 10 (1.00 g, 1.91 mmol) and a mixture of 5% Pd-C (300 mg ) was stirred vigorously at room temperature under an atmosphere of _{H 2.} After stirring overnight, the Pd-C catalyst was filtered off, and the filtrate was evaporated under reduced pressure to give crude amino acid (Compound 11) (719 mg, 97%). A solution of Fmoc-OSu (685 mg, 2.03 mmol) in dioxane (10 mL) is added to a stirred solution of compound 11 (719 mg, 1.85 mmol) and NaHCO ₃ (465 mg, 5.54 mmol) in water (30 mL), The solution was stirred at room temperature overnight. After dioxane removal, the solution was acidified with citric acid, extracted with EtOAc and dried over Na ₂ SO ₄ . Removal of solvent gave a white solid, which was purified by column chromatography on silica gel. The fraction eluted with 10% MeOH (in CHCl ₃ ) gave Fmoc-L-Lys [Boc, AEt (Boc)]-OH (Compound 12) (670 mg, 48%) as colorless crystals.
Mp 67-69 ° C .; [α] _D ²⁶ = +13.9 (c 0.86, CHCl ₃ ); IR (KBr) ₃ 3329, 3233, 2936, 2766, 2731, 2704, 2442, 1740, 1690, 1543, 1504, 1454 , 1273, 1169, 1038, 999 cm -1; 1 H NMR (400 MHz, CDCl 3) δ 8.57 (s, 1H), 7.74 (d, J = 7.6 Hz, 2H), 7.53-7.70 (m, 2H) , 7.37 (t, J = 7.4 Hz, 2 H), 7. 28 (t, J = 7.4 Hz, 2 H), 5.72-6. 13 (m, 1 H), 4.92-5. 23 (m, 1 H), 4.35-4. 46 (m, 3 H) ), 4.20 (t, J = 6.8 Hz, 1 H), 3.15-3.26 (m, 3 H), 1.25-1.91 (m, 6 H), 1.44 (s, 9 H), 1.42 (s, 9 H); ¹³ C NMR ( 100 MHz, CDCl ₃ ) δ 175.2, 156.4, 156.1, 143.8, 147.6, 127.0, 127.0, 125.1, 119.9, 80.1, 79.5, 53.6, 47.0, 46.2, 36.2, 39.3, 31.6, 28.3, 27.5, 22.2; FAB (+) HRMS calcd for C ₃₃ H ₄₆ N ₃ O ₈ [M ⁺ + H]: 612.3285; found: 612.3292.

A peptide having the following structure was synthesized in the same manner as in Example 2 using the obtained Fmoc-L-Lys [Boc, AEt (Boc)]-OH. The resulting labeled peptide may also be abbreviated as TMR-Gly- [L-Lys (AEt)] ₉ -NH ₂ (peptide 3) below.
The synthesized peptide was then purified using RP-HPLC. The homogeneity of the purified peptide was verified by analytical RP-HPLC and MALDI-TOF / MS. The results are shown in FIG. 2C.

Comparative Example 2: Synthesis of peptide starting from Fmoc-L-Lys (Boc) -OH The same as Example 2 except that Fmoc-Gly-OH and Fmoc-L-Lys (Boc) -OH were used as Fmoc amino acids. The peptide having the following structure was synthesized. The resulting labeled peptide may also be abbreviated as TMR-Gly- (L-Lys) ₉ -NH ₂ (peptide 1) hereinafter.
The synthesized peptide was then purified using RP-HPLC. The homogeneity of the purified peptide was verified by analytical RP-HPLC and MALDI-TOF / MS. The results are shown in FIG. 2A.

Comparative Example 3: Synthesis of a peptide starting from Fmoc-L-Arg (Pbf) -OH As in Example 2 except that Fmoc-Gly-OH and Fmoc-L-Arg (Pbf) -OH were used as Fmoc amino acids. The peptide having the following structure was synthesized. The resulting labeled peptide may be hereinafter also abbreviated as TMR-Gly- (L-Arg) ₉ -NH ₂ (peptide 2).
The synthesized peptide was then purified using RP-HPLC. The homogeneity of the purified peptide was verified by analytical RP-HPLC and MALDI-TOF / MS. The results are shown in FIG. 2B.

Comparative Example 4 Synthesis of a peptide using Fmoc-L-Arg (Pbf) -OH as a raw material The same as Comparative Example 3 except that 5 (6) -carboxyfluorescein was used instead of 5 (6) -tetramethylrhodamine carboxylic acid. Then, carboxyfluorescein (CF) labeled Arg peptide was synthesized. The resulting labeled peptide may hereinafter be abbreviated also as CF-Gly- (L-Arg) ₉ -NH ₂ (peptide 5).

Comparative Example 5 Synthesis of 1-ethylamino-2-guanidinylethane hydrochloride The title compound was synthesized as a model compound.

[1] 1- [N ′, N ′ ′-bis- (tert-butoxycarbonyl) -guanidinyl] -2- [N ′ ′ ′-(tert-butoxycarbonyl) -N ′ ′ ′-ethylamino] ethane ( Synthesis of Compound 21) N-ethylethylenediamine (compound 19) (205 mg, 2.32 mmol), 1,3-bis (tert-butoxycarbonyl) -2- (trifluoromethanesulfonyl) guanidine (1000 mg, 2.56 mmol) and It was added to a stirred solution of Et ₃ N (259 mg, 2.32 mmol) in CH ₂ Cl ₂ (20 mL) and stirred overnight at room temperature. The solvent was removed to give a residue, which was quickly purified by short column chromatography on silica gel (8% MeOH in CHCl ₃ ) to give compound 20 (768 mg, quant.). Add Boc ₂ O (760 mg, 3.49 mmol) and Et ₃ N (353 mg, 3.49 mmol) to a stirred solution of compound 20 (768 mg, 2.32 mmol) in CH ₂ Cl ₂ (30 mL) and stir for 2 hours at room temperature It stirred. Removal of solvent gave a residue, which was purified by column chromatography on silica gel (in 10% EtOAc in hexane) to give compound 21 (654 mg, 65%) as colorless crystals.
Mp 102-103 ° C .; IR (KBr) 332 3321, 3136, 2978, 2932, 1813, 1684, 1628, 1562, 1477, 1420, 1362, 1319, 1288, 1254, 1157 cm ^-1 ; ¹ H NMR ( 400 MHz, CDCl ₃ ) δ 11.32 (br s, 1H), 8.23-8.29 (m, 1H), 3.33-3.36 (m, 2H), 3.19-3.23 (m, 2H), 3.00-3.10 (m, 2H) , 1.32 (s, 9H), 1.28 (s, 9H), 1.25 (s, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 163.5, 156.3, 152.9, 82.9, 79.7, 79.1, 45.2, 41.8, 39.6 , 28.2, 28.0, 27.4, 13.4 ; FAB (+) HRMS calcd for C 20 H 39 N 4 O 6 [M + + H]: 431.2870; found: 431.2870.

[2] Synthesis of 1-ethylamino-2-guanidinylethane hydrochloride (compound 22) A solution of compound 21 (568 mg, 1.72 mmol) in 4 M HCl / dioxane (5 mL) was stirred at room temperature overnight. Solvent removal gave compound 22 (299 mg, 86%) as colorless crystals.
Mp 67-68 ° C .; IR (KBr) 3 3470, 3150, 2980, 2810, 2460, 2360, 2190, 2060, 1650, 1440 cm ^-1 ; ¹ H NMR (400 MHz, CDCl ₃ ) δ 3.56 (t, J = 6.2 Hz, 2 H), 3.19 (t, J = 6.1 Hz, 2 H), 3.08 (q, J = 7.3 Hz, 2 H), 1.31 (t, J = 7.3 Hz, 3 H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 158.9, 47.0, 44.5, 39.1, 11.5; FAB (+) HRMS calcd for C ₅ H ₁₅ N ₄ [M ⁺ + H]: 131.1275; found: 131.1300.

The structures of peptides 1 to 4 and model compounds 19 and 22 obtained in Examples and Comparative Examples are shown in FIG.
Test Example 1: Degree of protonation in GEt amine structure and AEt amine structure (Materials and Methods)
Compound 22 (comparative example 2), which is a model compound for Lys (GEt), and model compound 19 for Lys (AEt) were used.
Potentiometric titration N-ethylethylenediamine hydrochloride (compound 19 • 2 HCl; 80.5 mg, 0.5 mmol) and 1-ethylamino-2-guanidinylethane hydrochloride (compound 22; 101.5 mg, 0.5 mmol) were separately separated into 100 mM HCl ( It was dissolved in 10 mL to obtain a 100 mM amine and / or guanidine solution. Thereafter, potentiometric titration was performed at 20 ° C. using 100 mM NaOH. The titration was performed using a pH meter D-52 (Horiba, Kyoto, Japan). In this experiment, after the pH value stabilized, a 100 μL aliquot of titrant was added.
(result)
Compound 19 exhibited a typical two-step protonation reaction (α = 0.97 at pH 5.5, α = 0.66 at pH 7.4; pK _a1 7.1, pK _a2 10.3 ) (Fig. 3, ○). On the other hand, Compound 22 showed the same typical two-step protonation reaction although it was shifted to the high pH side (α = 0.99 at pH 5.5, α = 0.96 at pH 7.4. PK _a1 8.8, pKa ₂ 12.0) (FIG. 3, ●). From these results, it was found that the GEt amine structure can have a diprotonated structure even at pH 7.4 near neutral.

Test Example 2: Cellular Uptake of Various Peptides (Materials and Methods)
Huh-7 cells, HeLa cells or CHO-K1 cells were seeded in 24-well culture plates (40,000 cells per well) and incubated in 400 μl DMEM containing 10% fetal bovine serum (FBS). Thereafter, the medium was replaced with fresh medium containing 10% FBS, and each peptide solution was added to each well at a predetermined concentration (Fig. 4a, c and e). After 2 hours of incubation, the media was removed and the cells were supplemented with heparin (20 units / mL), trypsinized and washed with ice cold PBS. After adding the culture solution containing 10% FBS, the cells were centrifuged at 1600 rpm for 3 minutes at 4 ° C. The resulting cell pellet was suspended in ice cold PBS supplemented with heparin and centrifuged at 1600 rpm for 3 minutes at 4 ° C. Thereafter, it was treated with a cell lysis buffer M. The fluorescence intensity of each lysate was measured using a fluorometer (ND-3300, NanoDrop, Wilmington, DE). The amount of protein in each well was simultaneously measured using a BCA protein analysis reagent kit. The results are shown as the mean and standard deviation obtained from 3 samples.
In addition, Huh-7 cells and HeLa cells were used to keep the concentration of the peptide constant at 1 μM, while examining the cellular uptake of various peptides in the same manner for a predetermined incubation time (Fig. 5a and b).
(result)
Uptake of cellular peptides 1-4 into Huh-7 cells (Figure 4a), HeLa cells (Figure 4c) and CHO-K1 cells (Figure 4e) was evaluated at different concentrations. Peptide 2 showed the most efficient cellular uptake at concentrations of 1 μM (Huh-7 and HeLa cells) or 2 μM (CHO-K1 cells) and higher. On the other hand, at lower concentrations of 0.5, 0.25 and 0.125 μM, the uptake of peptide 4 was significantly higher than the uptake of the other peptides (eg P <0.05, peptide 2 vs. peptide 4, peptide concentration: 0.5 μM, Figure 4a). Cellular peptide 1, 2 and 3 uptake was undetectable at concentrations less than 0.5 and less than 0.5 and less than 1 μM, respectively.
Also, the order of intracellular uptake by Huh-7 cells and HeLa cells at a peptide concentration of 1 μM was the same at all incubation times (FIG. 5).

Test Example 3: Cytotoxicity of Various Peptides (Materials and Methods)
Huh-7 cells, HeLa cells or CHO-K1 cells were seeded into 96-well culture plates (10,000 cells / well) and incubated in 100 μL of DMEM containing 10% FBS. Thereafter, the medium was replaced with fresh medium containing 10% FBS, and each peptide solution was added to each well at a predetermined concentration. After 2 hours of incubation, measurements were made using cell-counting kit-8 according to the product protocol. Cell viability was assessed based on the absorbance of formazan from each well. Here, the cell viability was calculated with the absorbance of the wells containing no peptide as 100%. The results are presented as the mean and standard deviation obtained from 5 samples.
(result)
On all cells, peptide 4 showed slight cytotoxicity at high concentrations (Fig. 4b, d and f). Peptide 4 having a diprotonated GEt amine structure is compared to peptide 1 having a protonated primary amine structure, peptide 2 having a protonated guanidine structure and peptide 3 having a monoprotonated ethylene diamine structure It appears to be strongly bound to the cell membrane, which was thought to be responsible for efficient intracellular uptake at low concentrations and increased cytotoxicity at high concentrations.

Test Example 4: Effect of endosidesis inhibitor on intracellular uptake of various peptides In order to clarify the intracellular uptake pathway of peptide 2 and peptide 4 into Huh-7 cells, a specific endocytosis inhibitor (Amirolide being a macropinocytosis inhibitor: Amiloride. Ch: chlorpromazine and Su: sucrose being a clathrin-mediated endocytosis inhibitor. Ny: nystatin and Fi: Philippines being a caveola-mediated endocytosis inhibitor) The intracellular uptake experiment was performed using
(Materials and Methods)
Huh-7 cells were seeded in 24-well culture plates (40,000 cells / well) and incubated in 400 μL of DMEM containing 10% FBS. Fresh medium with 10% FBS, with or without amiloride (5 mM), chlorpromazine (10 μg / mL), sucrose (0.4 M), nystatin (25 μg / mL) or filipin (5 μg / mL) After medium change, cells were preincubated at 37 ° C. for 30 minutes. The peptide solution was added to each well at a concentration of 1 μM. After 2 hours of incubation, the media was removed and the cells were supplemented with heparin (20 units / mL), trypsinized and washed with ice cold PBS. After adding the culture solution containing 10% FBS, the cells were centrifuged at 1600 rpm for 3 minutes at 4 ° C. The resulting cell pellet was suspended in ice cold PBS supplemented with heparin and centrifuged at 1600 rpm for 3 minutes at 4 ° C. Thereafter, it was treated with a cell lysis buffer M. The fluorescence intensity of each lysate was measured using a fluorometer (ND-3300). The amount of protein in each well was simultaneously measured using a BCA protein analysis reagent kit. The results are presented as mean and standard deviation obtained from 3 samples.
(result)
The results are shown in FIG. FIG. 6a shows the uptake of peptide 2 in the presence of inhibitor. Am treatment reduced intracellular uptake by more than 30% (P <0.01). Also, Ch and Su significantly reduced the uptake of peptide 2 (P <0.05). These results indicate that peptide 2 is mainly macropinocytosis and is partially taken up by clathrin-mediated endocytosis. As shown in FIG. 6b, the uptake of peptide 4 in cells is approximately 30% lower (P <0.01) in cells treated with Ch and Su compared to controls. Incubation in the presence of Am significantly affected cellular uptake (approximately 15% reduction, P <0.05). These results indicate that peptide 4 is mainly clathrin-mediated endocytosis and is partially incorporated by macropinocytosis. Treatment of Huh-7 cells with Ny and Fi did not significantly affect uptake of either peptide 2 or 4. Thus, a slight difference was observed between the intracellular uptake pathways of peptides 2 and 4.

Test Example 5 Analysis of Intracellular Incorporation Mechanism of Peptide by Confocal Laser Scanning Microscopy In order to gain further insight into the different intracellular uptake mechanisms of peptide 4, using confocal laser scanning microscopy (CLSM) The intracellular distribution of peptides 2 and 4 was examined. CLSM observations of Huh-7 cells treated with TMR labeled peptide 2 or peptide 4 with CF labeled peptide 5 provided direct information on their different subcellular distribution.
(Materials and Methods)
Huh-7 cells or HeLa cells were seeded (20,000 cells / well) on an 8-well cover glass chamber (Iwaki, Tokyo, Japan) and incubated overnight in 200 μL of DMEM containing 10% FBS. The medium was then replaced with fresh medium containing 10% FBS. Peptide 2 or 4 and peptide 5 were added to each well at a concentration of 1 μM. After 2 hours of incubation, the medium was removed and heparin (20 units / mL) was added to the cells and washed three times with ice cold PBS. After nuclear staining with Hoechst 33342, subcellular distribution was observed by CLSM. CLSM observations are as follows: Excitation wavelength 405 nm (UV laser) for Hoechst 33342, Excitation wavelength 488 nm (Ar laser) for peptide 5 and Excitation wavelength 543 nm (He-Ne laser) for peptides 2 and 4, Plan -Apochromat 63x / 1.4 (Carl Zeiss) with LSM 710 (Carl Zeiss, Oberlochen, Germany) equipped with an objective lens. The proportion of peptide 2 or 4 colocalized with peptide 5 was quantified. The rate of colocalization was quantified as follows.

Here, "peptide pixel (co-localization)" indicates the number of pixels of peptide 2 or 4 co-localized with peptide 5 in the cell, and "peptide pixel (total)" indicates the total number of pixels in the cell. Show. The results are shown as mean and standard deviation obtained from 20 cells.
Furthermore, CLSM observation using Litho tracker green was performed as follows.
Huh-7 cells were seeded (20,000 cells / well) on 8-well cover glass chambers (Iwaki) and incubated overnight in 200 μL DMEM containing 10% FBS. Thereafter, the medium was replaced with fresh medium containing 10% FBS, and the peptide solution was added to each well at a concentration of 1 μM. After 2 hours of incubation, the medium was removed and the cells were supplemented with heparin (20 units / mL) and washed three times with ice cold PBS. Late endosomes / lysosomes were stained using Lysotracker green, nuclei were stained with Hoechst 33342, and subcellular distribution was observed by CLSM. CLSM observations are as follows: Plan-at excitation wavelength 405 nm (UV laser) for Hoechst 33342, excitation wavelength 488 nm (Ar laser) for litho tracker green, excitation wavelength 543 nm (He-Ne laser) for peptides 2 and 4 This was done using an LSM 710 (Carl Zeiss) equipped with an Apochromat 63x / 1.4 (Carl Zeiss) objective. The proportion of peptide 2 or 4 colocalized with Lysotracker green was quantified. The rate of colocalization was quantified as follows.

Here, “peptide pixel (co-localization)” indicates the number of pixels of peptide 2 or 4 co-localized with lyso tracker green in the cell, and “peptide pixel (total)” indicates all pixels in the cell. Indicates the number. The results are shown as the mean and standard deviation obtained from 18 cells.
(result)
The percentage of colocalization quantified was 70% of peptide 2 and 47% of peptide 4 co-localized with peptide 5 (FIG. 7a). A significant difference was observed between peptides 2 and 4 (P <0.001). This result suggested that the final destinations of peptides 2 and 4 in Huh-7 cells were different. HeLa cells and CHO-K1 cells treated with peptides 2 and 4 were also, along with peptide 5, 83% and 67% (HeLa cells, FIG. 7b), 87% and 75% (CHO-K1 cells, respectively). The colocalization rate of 7c) was shown (P <0.001).
However, CLSM observation of peptide 2 and peptide 4 with Lysotracker green staining revealed similar co-localization rates in acidic late endosomes / lysosomes (FIG. 8) .
From the above results, it was found that peptide 4 can bind to the cell membrane more strongly than peptide 2 and is more efficiently taken up into cells at low concentrations by a slightly different mechanism.

Test Example 6: Gene Transfer Using Plasmid DNA (pDNA) (Materials and Methods)
Using two types of Huh-7 cells and CHO-K1 cells, the amount of expressed protein was quantified (= gene transfer efficiency) using pDNA encoding luciferase protein. The commercially available gene transfer reagent TurboFect was used as a positive control. At the same time, the cytotoxicity was also examined in the same manner as in Test Example 3.
(result)
Peptide 4 showed high gene transfer efficiency at low concentration, and in Huh-7 cells, it showed efficiency similar to that of TuboFect.
The lower part of FIG. 9 shows the results of evaluation of cytotoxicity under the same conditions. Although peptide 4 is slightly toxic when used at high concentrations, TurboFect is quite toxic at this concentration.
From these results, it was shown that toxicity is not a problem in the concentration range where the peptide of the present invention exhibits high gene transfer efficiency.

  The peptide synthesized from the non-natural amino acid of the present invention having a guanidinylalkylamino group which is a cationic functional group in the side chain has lower concentration as a drug delivery carrier as compared with a conventional membrane permeable peptide It can exert its function. Therefore, it can be used as a drug delivery carrier without side effects such as toxicity. The present invention makes it possible to develop reagents that introduce proteins such as antibodies and genes such as plasmid DNA and siRNA into cells and safer and more effective pharmaceuticals.

Claims (14)

Formula (I):

Wherein, _{n 1} represents an integer of 1 to 3; _{n 2} represents an integer of ^1~6; R 1 ~R ³ is the same or different, a protecting group of a hydrogen atom or an amino group; ^{R 4} Is a hydrogen atom, a protecting group of an amino group, an amino acid residue or an acetyl group; R ⁵ is an amino acid residue, -NR ₂ , -OR or -SR (wherein R is a hydrogen atom or 1 to 6 carbon atoms compounds represented by a 'straight represents a chain or branched chain alkyl group), wherein the protecting group of the amino group 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc ), 3-nitro-2-pyridinesulfenyl (Npys), benzyloxycarbonyl (Cbz), dansyl (DNS), isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, chloroformate ben It is selected from benzyl (Cl-Cbz), benzyl bromoformate (Br-Cbz), adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl and diphenylphosphinothiol . The compound according to claim 1, wherein R ^{1 to} R ³ are the same or different and are protecting groups of amino group. The compound of claim 1, wherein R ^{1 to} R ³ are hydrogen atoms.   An oligopeptide consisting of 5 to 15 amino acid residues, wherein at least 20% or more of the total amino acid residues of the peptide are derived from the compound according to any one of claims 1 to 3. Oligopeptide. At least 20% or more of all amino acid residues are amino acid residues of the formula (I '):

(Wherein each symbol has the same meaning as in claim 1)
The oligopeptide of Claim 4 which is derived from the compound represented by these. Formula (II):
(Xaa) n (II)
[Wherein, n is any integer of 5 to 15; n Xaa's are the same or different and any amino acid residue]
An oligopeptide represented by
At least one in the case of n = 5, at least two in the case of n = 6 to 10, and at least three Xaa in the case of n = 11 to 15:

(Wherein, n ₁ represents an integer of ₁ to 3; n ₂ represents an integer of 1 to 6; R ^{1 to} R ³ are the same or different and are a hydrogen atom or a protecting group of an amino group; * represents Show binding site)
Wherein the protective group of the amino group is 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), 3-nitro-2-pyridinesul Phenyl (Npys), benzyloxycarbonyl (Cbz), dansyl (DNS), isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, benzyl chloroformate (Cl-Cbz), benzyl bromoformate (Br-Cbz), adamantyloxy It is selected from carbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl and diphenylphosphinothiol .   A compound formed by combining the oligopeptide according to any one of claims 4 to 6 with a molecule X (X represents a physiologically active substance or a labeling compound).   A composition comprising the oligopeptide according to any one of claims 4 to 6 and a molecule X (X represents a physiologically active substance or a labeling compound).   The compound according to claim 7, wherein X is a physiologically active substance.   The composition according to claim 8, wherein X is a physiologically active substance.   A pharmaceutical composition comprising a compound according to claim 9 and / or a composition according to claim 10.   The compound according to claim 7, wherein X is a labeling compound.   The composition according to claim 8, wherein X is a labeling compound. A cell labeling reagent comprising the compound according to claim 12 and / or the composition according to claim 13 .