WO2022149584A1 - Peptide - Google Patents

Abstract

The present invention provides a peptide having a fluoroalkyl group as a side chain. The present invention pertains to a peptide in which two or more amino acids are peptide-bonded, wherein at least one of the amino acid residues constituting the peptide has, as a side chain, a C_1-30 alkyl group substituted with at least two fluorine atoms or a group having 1 to 5 ether-bonding oxygen atoms between carbon atoms in a C_2-30 alkyl group substituted with at least two fluorine atoms.

Description

peptide

The present invention relates to peptides containing amino acid residues in which a fluoroalkyl group has been introduced into the side chain.
This application claims priority based on Japanese Patent Application No. 2021-001145 filed in Japan on January 6, 2021, and the contents thereof are incorporated herein by reference.

Antibody drugs, peptide drugs, nucleic acid drugs, etc. have the advantages of high specificity for target molecules and few side effects. However, all of them have a problem that it is difficult to reach the target molecule existing in the cell. Various methods are being studied to solve this problem. Among them, cell membrane penetrating peptides (CPP) are expected to be promising. Typical examples of the CPP include a peptide derived from the TAT protein of the HIV virus (Patent Document 1) and a peptide having a polyArg sequence (Patent Document 2). This can be combined with the medicinal peptide to transport the medicinal peptide into the cell (for example, Patent Document 3 and Non-Patent Document 1).

On the other hand, fluorine-containing amino acids have been reported to exhibit unique physiological activity and are attracting attention. For example, 3,3,3-trifluoroalanine and its derivatives have been reported to act as a suicide inhibitor of pyridoxal enzyme (Non-Patent Document 2). In addition, it has been reported that alanine racemase of Gram-negative bacterium Salmonella typhimurium and Gram-positive bacterium Bacillus stearothermophilus is inactivated by 3,3,3-trifluoroalanine (Non-Patent Document 3). Fluorine-containing amino acids and peptides containing them are expected to be used in the pharmaceutical field as physiologically active substances.

It is known that a compound having a polyfluoro structure is stable and low in toxicity in vivo, and is excellent in intracellular uptake and escape from endosomes (Non-Patent Document 4). It has been reported that a peptide dendrimer using lysine in which a side chain amino group is perfluoroacylated can be used for gene delivery by utilizing this property (Non-Patent Document 5). However, since it is a dendrimer, it cannot form a hybrid that is bound to a medicinal active peptide, nucleic acid, or protein that is an antibody drug like CPP.

US Pat. No. 6,316,003 US Pat. No. 6,306,993 International Publication No. 2008/08491

The CPP described in Patent Document 1 and the like has various problems such as intracellular transport efficiency and decomposition by peptidase in a living body.
An object of the present invention is to provide a peptide containing an amino acid residue having a fluoroalkyl group introduced into a side chain and a method for producing the same.

The present inventors have produced a peptide containing an amino acid residue having a fluoroalkyl group introduced into the side chain, and found that the peptide has excellent cell membrane permeability, and completed the present invention.

That is, the present invention is as follows.
[1] A peptide in which two or more amino acids are peptide-bonded.
At least one of the amino acid residues constituting the peptide is C _1-30 alkyl group substituted with at least two fluorine atoms in the side chain, or C _2- replaced with at least two fluorine atoms. A peptide having a group having 1 to 5 ether-bonding oxygen atoms between carbon atoms of a ₃₀ alkyl group.
[2] 1 to 5 ether bonds between the carbon atoms of the C _1-30 alkyl group substituted with at least two fluorine atoms or the C _2-30 alkyl group substituted with at least two fluorine atoms. The peptide having an oxygen atom of [1] may be further substituted with a halogen atom other than the fluorine atom.
[3] 1 to 5 ether bonds between the carbon atoms of the C _1-30 alkyl group substituted with at least two fluorine atoms or the C _2-30 alkyl group substituted with at least two fluorine atoms. The side chain having a group having an oxygen atom of is the following general formula (f-1) or (f-2).

(In the formula, Rf ^P is between carbon atoms of a fully halogenated C _1-10 alkyl group containing at least two or more fluorine atoms or a fully halogenated C _2-10 alkyl group containing at least two or more fluorine atoms. Represents a group having 1 to 5 ether-bonding oxygen atoms, n1 is an integer of 0 to 10, n2 is an integer of 0 to 9, and a black circle means a bond).
The peptide of the above [1] or [2], which is a group represented by.
[4] The peptide according to any one of [1] to [3] above, wherein the C-terminal or N-terminal may be protected by a protecting group.
[5] The peptide according to any one of the above [1] to [4], which is permeable to cell membranes.

The peptide according to the present invention has excellent cell membrane permeability because a fluoroalkyl group is introduced into the side chain. Therefore, the peptide is expected to be used in the pharmaceutical field as a physiologically active substance.

In Test Example 1, peptide fluorescence conjugate 1 (Alexa-Ala-[(R) -RFAA (C8)]-Phe-OMe, diastereomer A) (PFCJ1), peptide fluorescence conjugate 2 (Alexa-Ala- [ (S) -RFAA (C8)]-Phe-OMe, Diasteromer B) (PFCJ2), Peptide Fluorescence Conjugate 5 (Alexa-Ala-RFAA (C4) -Phe-OMe) (PFCJ5), Peptide Fluorescence Conjugate HeLa cells treated at 37 ° C. for 1 hour in a sample solution containing 6 (Alexa-Ala-Nle-Phe-OMe) (PFCJ6) or fluorescent dye 1 (diethylamide of the fluorescent substance AlexaFluoro647) (FD1). It is a figure which showed the result of flow cytometry. In Test Example 1, HeLa cells treated at 37 ° C. for 1 hour in a sample solution containing peptide fluorescence conjugate 1, peptide fluorescence conjugate 2, peptide fluorescence conjugate 5, peptide fluorescence conjugate 6, or fluorescent dye 1 It is a figure which compared the average fluorescence intensity in flow cytometry. In Test Example 1, treatment at 37 ° C. for 1, 4, 24 hours in a sample solution containing peptide fluorescence conjugate 1, peptide fluorescence conjugate 2, peptide fluorescence conjugate 5, peptide fluorescence conjugate 6, or fluorescent dye 1. It is a figure which compared the time-dependent change of the average fluorescence intensity in the flow cytometry of the HeLa cell. In Test Example 1, HeLa treated at 37 ° C. for 1 hour in a sample solution containing peptide fluorescence conjugate 1, peptide fluorescence conjugate 2, or peptide fluorescence conjugate 3 (Alexa-RFAA (C6) -Phe-OMe). It is a figure which showed the result of the flow cytometry of a cell. In Test Example 1, the figure which showed the result of the flow cytometry of the HeLa cell treated at 37 degreeC for 4 hours in the sample solution containing peptide fluorescence conjugate 1, peptide fluorescence conjugate 2, or peptide fluorescence conjugate 3. be. In Test Example 1, the results of flow cytometry of HeLa cells treated at 4 ° C. for 40 minutes in a sample solution containing peptide fluorescent conjugate 1, peptide fluorescent conjugate 2, peptide fluorescent conjugate 3, or fluorescent dye 1 are shown. It is a figure shown. In Test Example 3, the peptide fluorescence conjugate 7 (Alexa-Ala-[(R) -RFAA (C6)]-Phe-OMe) and the peptide fluorescence conjugate 1 (Alexa-Ala-[(R) -RFAA (C8)) ] -Phe-OMe, Diasteromer A), Peptide Fluorescence Conjugate 2 (Alexa-Ala-[(S) -RFAA (C8)]-Phe-OMe, Diasteromer B), Peptide Fluorescence Conjugate 5 (Alexa) -Ala-RFAA (C4) -Phe-OMe), peptide fluorescent conjugate 6 (Alexa-Ala-Nle-Phe-OMe), or in a sample solution containing a diethylamide (fluorescent dye 1) of the fluorescent substance AlexaFluoro 647. , Is a diagram showing the results of flow cytometry of HeLa cells treated at 37 ° C. for 1 hour. In Test Example 3, in a sample solution containing a peptide fluorescence conjugate 7, a peptide fluorescence conjugate 1, a peptide fluorescence conjugate 2, a peptide fluorescence conjugate 5, a peptide fluorescence conjugate 6, or a fluorescent dye 1, 1 at 37 ° C. It is a figure which compared the average fluorescence intensity in the flow cytometry of the time-treated HeLa cells.

In the present invention and the present specification, "C _p1-p2 " (p1 and p2 are positive integers satisfying p1 <p2) means that the number of carbon atoms is a group of p1 to p2.

In the present invention and the present specification, the "C _1-10 alkyl group" is an alkyl group having 1 to 10 carbon atoms, and may be a straight chain or a branched chain. The "C _2-10 alkyl group" is an alkyl group having 2 to 10 carbon atoms, and may be a straight chain or a branched chain. Examples of C _1-10 alkyl groups are methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert- Examples thereof include a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group and the like.

In the present invention and the present specification, the "C _1-30 alkyl group" is an alkyl group having 1 to 30 carbon atoms, and may be a straight chain or a branched chain. The "C _2-30 alkyl group" is an alkyl group having 2 to 30 carbon atoms, and may be a straight chain or a branched chain. Examples of C _1-30 alkyl groups are methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert- Pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group, heneikosyl group. , Docosyl group, tricosyl group, tetracosyl group, pentacosyl group, hexacosyl group, heptacosyl group, octacosyl group, nonacosyl group, triacontyl group and the like.

In the present invention and the present specification, the "C _1-6 alkyl group" is an alkyl group having 1 to 6 carbon atoms, and may be a straight chain or a branched chain. Examples of C _1-6 alkyl groups are methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert- Examples thereof include a pentyl group and a hexyl group.

In the present invention and the present specification, the "C _6-14 aryl group" is an aromatic hydrocarbon group having 6 to 14 carbon atoms, and a C _6-12 aryl group is particularly preferable. Examples of the C _6-14 aryl group include a phenyl group, a naphthyl group, an anthryl group, a 9-fluorenyl group and the like, and a phenyl group is particularly preferable.

In the present invention and the present specification, the "optionally substituted C _6-14 aryl group" is one or more hydrogen atoms bonded to the carbon atom of the C _6-14 aryl group, preferably 1 to 1. Three are groups substituted with other functional groups. When having two or more substituents, the substituents may be the same kind or different from each other. The substituents include a nitro group, a halogen atom (fluorine atom, chlorine atom, bromine atom, or iodine atom), a C _1-6 alkyl group, a C _1-6 alkoxy group, and a methylenedioxy group (-O-CH). ₂ -O-) and the like can be mentioned. Examples of "optionally substituted C _6-14 aryl groups" are phenyl group, naphthyl group, anthryl group, 4-nitrophenyl group, 4-methoxyphenyl group, 2,4-dimethoxyphenyl group, 3, Examples thereof include 4-dimethoxyphenyl group, 4-methylphenyl group, 2,6-dimethylphenyl group, 3-chlorophenyl group, 1,3-benzodioxol-5-yl group and the like.

In the present invention and the present specification, the "C _6-14 aryl-C _1-6 alkyl group" is a C _6-14 aryl group in which one hydrogen atom bonded to the carbon atom of the C _1-6 alkyl group is used. It is a group substituted with. Examples of the C _{6-14 aryl group in the C 6-14 aryl} -C _1-6 alkyl group include a phenyl group, a naphthyl group, an anthryl group, a 9-fluorenyl group and the like, and a phenyl group or a 9-fluorenyl group is particularly preferable. .. As the C _1-6 alkyl group in the C _6-14 aryl-C _1-6 alkyl group, a C _1-4 alkyl group is preferable. Examples of the C _6-14 aryl-C _1-6 alkyl group include a benzyl group, a diphenylmethyl group, a triphenylmethyl group, a 2-phenylethyl group, a 9-anthrylmethyl group, a 9-fluorenylmethyl group and the like. Can be mentioned.

In the present invention and the present specification, the "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. The "halogen atom other than the fluorine atom" means a chlorine atom, a bromine atom, or an iodine atom. As an example of the "halogen atom other than the fluorine atom", a chlorine atom or a bromine atom is preferable, and a chlorine atom is particularly preferable.

In the present invention and the present specification, the "C _1-6 alkoxy group" means a group in which an oxygen atom is bonded to the bond end of a C _1-6 alkyl group having 1 to 6 carbon atoms. The C _1-6 alkoxy group may be a straight chain or a branched chain. Examples of the C _1-6 alkoxy group include a methoxy group, an ethoxy group, a propoxy group, a butoxy group, a tert-butoxy group, a pentyloxy group, a hexyloxy group and the like.

In the present invention and the present specification, the "ether-bonded oxygen atom" is an oxygen atom that connects carbon atoms, and does not include an oxygen atom in which oxygen atoms are connected in series. The maximum number of ether-bonding oxygen atoms that an alkyl group having Nc carbon atoms (Nc is an integer of 2 or more) can have is Nc-1.

Further, hereinafter, "compound n" means a compound represented by the formula (n).

<Fluoroalkyl group-containing peptide>
The peptide according to the present invention is a peptide consisting of two or more amino acids, and at least one of the amino acid residues constituting the peptide is C _1- in which at least one of the amino acid residues is substituted in the side chain with at least two fluorine atoms. It has ₃₀ alkyl groups. When the C _1-30 alkyl group consists of two or more carbon atoms (in the case of a C _2-30 alkyl group), even if it has 1 to 5 ether-bonding oxygen atoms between the carbon atoms. good. In the present invention and the present specification, "C _1-30 alkyl group substituted with at least two fluorine atoms (the C _1-30 alkyl group is 1 to 5 between carbon atoms when the number of carbon atoms is 2 or more). It may have an ether-bonding oxygen atom) "means" a C _1-30 alkyl group substituted with at least two fluorine atoms, or a C substituted with at least two fluorine atoms. It means "a group having 1 to 5 ether-bonding oxygen atoms between carbon atoms of a _2-30 alkyl group".
In the present invention and the present specification, "a peptide having a C _1-30 alkyl group substituted with at least two fluorine atoms in the side chain" may be referred to as "fluoroalkyl group-containing peptide". Hereinafter, "a group in which at least two hydrogen atoms bonded to a carbon atom of the C _1-30 alkyl group are substituted with a fluorine atom" may be referred to as "Rf".

In Rf, one or more hydrogen atoms bonded to carbon atoms may be further substituted with halogen atoms other than fluorine atoms. Here, as the C _1-30 alkyl group of Rf, a C _1-20 alkyl group is preferable, a C _1-10 alkyl group is more preferable, a C _2-10 alkyl group is further preferable, and a C _2-8 alkyl group is more preferable. Even more preferable. When the C1-30 alkyl group is a _C2-30 alkyl group, it may have 1 to 5 ether-bonding oxygen atoms between carbon atoms. In Rf, the number of hydrogen atoms substituted with fluorine atoms is not particularly limited as long as it is 2 or more, for example, 3 or more is preferable, 6 or more is more preferable, and 7 or more is further preferable. preferable.

Examples of Rf include trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, perfluorodecyl group, Difluoromethyl group, 1,1-difluoroethyl group, 2,2-difluoroethyl group, 1,1,2,2-tetrafluoroethyl group, 1,1,2,2,3,3-hexafluoropropyl group, 1,1,2,3,3,3-hexafluoropropyl group, 1,1,2,2,3,3-hexafluorohexyl group, 1,1,2,2,3,3-hexafluorooctyl group , 1,1,2,2,3,3-hexafluorodecyl group, 1,1,2,2,3,3-hexafluorooctadecyl group, 1,1,2,2,3,3-hexafluorohexa Examples include a cosyl group.

When Rf is a group having 2 carbon atoms, the Rf is bonded to a carbon atom like a pentafluoroethyl group rather than a 1,1,1-trifluoroethyl group (CF ₃ -CH _2- ). A group in which at least four or more hydrogen atoms are substituted with a fluorine atom is preferable. When Rf is a group having 3 carbon atoms, a linear group is preferable as Rf, and when it is a branched chain group, 1,1,1,3,3,3-hexafluoropropane. A group having 0 or 1 trifluoromethyl group is preferable to a group having 2 trifluoromethyl groups such as -2-yl group ((CF ₃ ) ₂ -CH-). When Rf is a group having 4 carbon atoms, a linear group is preferable as Rf, and in the case of a branched chain group, a hydrogen atom bonded to a carbon atom constituting an alkylene group portion becomes a fluorine atom. It is preferably a substituted group or a fully fluorinated group.

As Rf, a group represented by the following general formula (f-1) or (f-2) is preferable. Here, Rf ^P represents a fully halogenated C _1-10 alkyl group containing at least two or more fluorine atoms. Rf ^P is a group in which all the hydrogen atoms of the C _1-10 alkyl group are substituted with halogen atoms, and at least two or more of these halogen atoms are fluorine atoms. When Rf ^P has 2 or more carbon atoms, that is, when it is a fully halogenated C _2-10 alkyl group, it may have 1 to 5 ether-bonding oxygen atoms between carbon atoms. In the present invention and the present specification, "a fully halogenated C _1-10 alkyl group containing at least two or more fluorine atoms (the C _1-10 alkyl group is used between carbon atoms when the number of carbon atoms is two or more." It may have an ether-bonding oxygen atom) "means" a fully halogenated C _1-10 alkyl group containing at least two or more fluorine atoms, or a completely halogen containing at least two or more fluorine atoms. C _2-10 A group having 1 to 5 ether-bonding oxygen atoms between carbon atoms of an alkyl group. In the general formula (f-2), the two Rf ^Ps may be groups of the same kind or different groups from each other.

In the following general formula (f-1) or (f-2), n1 is an integer of 0 to 10, and n2 is an integer of 0 to 9. When n1 and n2 are 0, both represent a single bond. That is, when n1 is 0, the group represented by the general formula (f-1) is Rf ^P −, and when n2 is 0, the group represented by the general formula (f-2) is (Rf). ^P ) ₂ -CH-.

When Rf is a group represented by the general formula (f-1), Rf ^P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluoro. A hexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, preferably a group in which n1 is an integer of 0 to 4, and Rf ^P is a trifluoromethyl group, a pentafluoroethyl group, or a hepta. A fluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, and a group in which n1 is an integer of 0 to 2 is more preferable. Rf ^P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, and n1 is an integer of 0 to 2 (where n1 is). 1 and excluding the group in which Rf ^P is a trifluoromethyl group) is more preferable.

When Rf is a group represented by the general formula (f-2), Rf ^P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluoro. A hexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, preferably a group in which n2 is an integer of 0 to 4, and Rf ^P is a trifluoromethyl group, a pentafluoroethyl group, or a hepta. A fluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, and a group in which n2 is an integer of 0 to 2 is more preferable. Rf ^P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, and n2 is an integer of 0 to 2 (where n2 is). (Excluding the group which is 0 or 1 and Rf ^P is a trifluoromethyl group) is more preferable.

Examples of Rf are difluoromethyl group, 1,1-difluoroethyl group, 2,2-difluoroethyl group, 1,1,2,2-tetrafluoroethyl group, 1,1,2,2,3,3. -It may be a hexafluoropropyl group, a 1,1,2,3,3,3-hexafluoropropyl group or the like.

Examples of the fluoroalkyl group-containing peptide according to the present invention include peptides containing at least one amino acid residue having an Rf side chain. Of the amino acid residues constituting the peptide, at least one side chain may be Rf, and the side chains of all amino acid residues may be Rf. When a single molecule peptide has two or more amino acid residues having a side chain of Rf, these plurality of Rfs may be the same species or different from each other. Further, among the peptides, the amino acid residue whose side chain is Rf may be at the N-terminal, at the C-terminal, or at a position other than the C-terminal.

The fluoroalkyl group-containing peptide according to the present invention may be a peptide consisting of two or more amino acids, and a peptide consisting of three or more amino acids is also preferable. The fluoroalkyl group-containing peptide according to the present invention is preferably a peptide consisting of 2 to 40 amino acids, and more preferably a peptide consisting of 3 to 20 amino acids.

The C-terminus of the fluoroalkyl group-containing peptide according to the present invention may be protected by a protecting group. As the C-terminal protecting group, the group listed in R ¹ below can be used, and a benzyl group is preferable. Further, the N-terminal of the fluoroalkyl group-containing peptide according to the present invention may be protected by a protecting group of an amino group. As the N-terminal protecting group, the group listed in ^R2 below can be used, and a Boc group or an Fmoc group is preferable.

Among the fluoroalkyl group-containing peptides according to the present invention, the amino acid residue having no Rf in the side chain is not particularly limited, and may be an amino acid residue of α-amino acid, or β-amino acid. It may be an amino acid residue of γ-amino acid, an amino acid residue of δ-amino acid, or an amino acid residue of δ-amino acid. Further, it may be an amino acid residue of L-amino acid or an amino acid residue of D-amino acid. Amino acid residues that do not have Rf in the side chain contained in the fluoroalkyl group-containing peptide according to the present invention include amino acids constituting proteins, D-forms thereof, and modified amino acids in which these side chains are modified. It is preferably an amino acid residue.

Examples of amino acids constituting the protein include glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamine, proline, aspartic acid, glutamic acid, lysine, arginine, and histidine. Examples of the modified amino acid in which the amino acids constituting the protein are modified include the hydrogen atom of the amino group of the side chain of lysine, arginine, and histidine, the group mentioned in ^R2 below, and Pbf (N-ω- (2). , 2,4,6,7 ^- Pentamethyldihydrobenzofuran-5-sulfonyl) Amino acid substituted with a group; Amino acids substituted with an alkyl group; amino acids in which the hydrogen atom of the thiol group of cysteine is substituted with a benzyl group can be mentioned.

Examples of the fluoroalkyl group-containing peptide according to the present invention include tripeptides represented by the following general formula (101) or (102). In the general formulas (101) and (102), R ¹¹ and R ¹² are preferably C _1-6 alkyl groups or benzyl groups, respectively, and preferably methyl groups or benzyl groups, respectively. It is particularly preferred that R ¹¹ is a methyl group and R ¹² is a benzyl group. X is Fmoc or Boc. Z is a C _1-6 alkoxy group, and a methoxy group is particularly preferable.

In the general formulas (101) and (102), Rf ^P , n1 and n2 are the same as those in the general formulas (f-1) and (f-2). As the group represented by the general formula (101) or (102), Rf ^P is preferably a fully fluorinated C _1-10 alkyl group, and n1 or n2 is preferably an integer of 0 to 4, preferably Rf ^P. Is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, or a perfluorooctyl group, and n1 or n2 is an integer of 0 to 2. It is more preferable that Rf ^P is a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, or a perfluorooctyl group, and n1 or n2 is an integer of 0 to 2.

The fluoroalkyl group-containing peptide according to the present invention may be carried out by a general peptide synthesis method except that a fluoroalkyl group-containing amino acid, which is an amino acid having a fluoroalkyl group introduced into a side chain, is used as a raw material amino acid. can. For example, it can be carried out by a peptide solid phase synthesis method. The fluoroalkyl group-containing peptide can be easily synthesized using an automatic peptide synthesizer using an amino acid having a fluoroalkyl group introduced into the side chain as a raw material. As the fluoroalkyl group-containing amino acid, an amino acid having an Rf side chain is preferable.

A peptide can be produced by sequentially condensing an amino acid having an amino group protected with an amino acid having a C-terminal bonded to a solid phase and desorbing the peptide from the solid phase. As the amino acid raw material, it is preferable to use one in which the amino group is protected by a Boc group or an Fmoc group. As the side chain functional group of the amino acid raw material, it is preferable to use one protected by a protecting group. Examples of the protecting group of the side chain functional group include a Boc group, a triphenylmethyl group, a benzyl group, a 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) group and the like.

Examples of the condensing agent forming a peptide bond include N, N-dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide (WSC), and benzotriazole-1-yloxy-trisdimethyl. Aminophosphonium hexafluorophosphate (BOP), benzotriazole-1-yloxytrispyrrolidinophosphonium hexafluorophosphate (pyBOP), 2- (1H-benzotriazole-1-yl) -1,1,3 3-Tetramethyluronium hexafluorophosphate (HBTU), 2- (1H-benzotriazole-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate, 1-cyano-2- Ethoxy-2-oxoethylideneaminooxy) dimethylaminomorpholinocarbenium hexafluorophosphate (COMU) and the like can be mentioned. Further, N-hydroxybenzotriazole (HOBt), ethyl (hydroxyimino) cyanoacetate (oxyma) and the above condensing agent can be mixed and used in a preferable ratio.

A method of activating the carboxy terminal may be used for the formation of the peptide bond, and examples of the activator include N-hydroxysuccinimide, p-nitrophenyl ester, pentafluorophenyl ester and the like. Examples of the base used for forming a peptide bond include triethylamine, diisopropylethylamine (DIPEA) and the like. Examples of the solvent used for the peptide bond forming reaction include chloroform, dichloromethane, acetonitrile, N, N-dimethylformamide (DMF), dimethyl sulfoxide and the like.

The Boc and Fmoc groups, which are the protecting groups for the amino-terminal amino groups of peptides or amino acids, can be removed with trifluoroacetic acid or piperidine, respectively. The protecting group of the side chain functional group of the amino acid residue of the peptide can be removed by, for example, trifluoroacetic acid, hydrogen fluoride (HF), trifluoromethanesulfonic acid or the like.

Further, in the peptide solid phase synthesis method, for example, TFA can be used as a method for removing a peptide having a protecting group on the side chain functional group of the peptide or amino acid residue from the peptide solid phase synthetic resin. Desorption of the peptide from the peptide solid phase resin and desorption of the protecting group of the side chain functional group of the amino acid residue can also be performed simultaneously in the same reaction system. Alternatively, each can be done independently. Examples of the peptide solid phase synthetic resin for peptide solid phase synthesis include 4-hydroxymethyl-3-methoxyphenoxybutyic acid-benzhydrylamine-polystyrene resin, p-benzyloxybenzyl alcohol-polystyrene resin, and oxime resin, which are usually commercially available. Can be used.

The target peptide or its intermediate is isolated and purified by various methods such as ion chromatography, gel filtration chromatography, reverse phase chromatography, normal phase chromatography, recrystallization, extraction, and fractional crystallization. be able to. In addition, the peptide thus obtained can be converted into each salt by a conventional method.

The protecting group of the amino group or the carboxy group of the produced fluoroalkyl group-containing peptide can also be deprotected if necessary. Deprotection can be performed by a conventional method depending on the type of protecting group.

<Synthetic reaction of amino acid containing fluoroalkyl group>
The fluoroalkyl group-containing amino acid can be produced, for example, by the following synthetic reaction.

Specifically, as Rf, a group represented by the general formula (f-1) or (f-2) described later is preferable.

R ¹ is a protecting group for a carboxy group, and specifically, a group represented by the following general formula (p-1), a 2- (9,10-dioxo) anthrylmethyl group, a benzyloxymethyl group, and the like. And a protecting group selected from phenacyl groups. In the general formula (p-1), R ³ is a optionally substituted C _6-14 aryl group, and R ⁴ and R ⁵ are independently hydrogen atoms or optionally substituted C. It is a _6-14 aryl group. The black circle means a bond.

Benzyl group, diphenylmethyl group, triphenylmethyl group, 4 ^- nitrobenzyl group, 4-methoxybenzyl group, 2,4-dimethoxybenzyl group, 3,4- Dimethoxybenzyl group, 4-methylbenzyl group, 2,6-dimethylbenzyl group, 3-chlorobenzyl group, 9-anthrylmethyl group, piperonyl group, 2- (9,10-dioxo) anthrylmethyl group, benzyloxy Examples thereof include a methyl group and a phenylyl group. R ¹ is preferably a benzyl group, a triphenylmethyl group, and more preferably a benzyl group in that it can be deprotected under mild conditions.

In the production method, R1 can be deprotected under mild conditions by using an aralkyl protecting group such as a benzyl group or a triphenylmethyl group as the protecting group ^R1 of the carboxy group, and the functional group of the amino acid is decomposed. It is advantageous in that it is possible to synthesize a fluorine-containing amino acid and a fluorine-containing peptide.

^R6 is a silyl protecting group. Examples of R ⁶ include a trimethylsilyl (TMS) group, a triethylsilyl (TES) group, a triisopropylsilyl (TIPS) group, a tert-butyldimethylsilyl (TBDMS) group, a tert-butyldiphenylsilyl (TBDPS) group and the like. Preferably, ^R6 is a trimethylsilyl (TMS) group.

R ² is a protecting group for the amino group. ^R2 is not particularly limited as long as it is a protecting group for an amino group used in peptide synthesis. As the protective group of the amino group, tert-butoxycarbonyl (Boc) group, 9-fluorenylmethyloxycarbonyl (Fmoc) group, benzyloxycarbonyl (Cbz) group, allyloxycarbonyl (Allloc) group, 2,2 Examples thereof include a carbamate-based protective group such as a 2-trichloroethoxycarbonyl (Troc) group. R2 is preferably a tert-butoxycarbonyl (Boc) group or a 9-fluorenylmethyloxycarbonyl (Fmoc) group in that it can be deprotected under mild conditions.

[Step 1]
Compound 2-2 can be obtained by reacting compound 2 and compound 8 in the presence of metal fluoride. Since the compound 8 represented by the general formula (8) Rf-R6 can be synthesized from easily available Rf-I (fluoroalkyl iodide) in one step, the range of Rf groups that can be introduced is wide.

As the metal fluoride, alkali metal fluorides such as cesium fluoride, lithium fluoride and sodium fluoride can be used, and cesium fluoride is preferable.
The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include inert solvents such as tetrahydrofuran (THF), dichloromethane (DCM), acetonitrile, benzene, toluene, diethyl ether, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide and the like. , Tetrahydrofuran is preferred.

The amount of compound 8 is preferably 0.5 to 10 mol with respect to 1 mol of compound 2. The amount of metal fluoride is preferably 0.01 to 2 mol with respect to 1 mol of compound 2. The reaction in step 1 is preferably carried out at a temperature of 10 ° C. or lower. By carrying out the reaction at a temperature of 10 ° C. or lower, compound 2-2 can be produced in high yield. The reaction temperature is preferably −78 ° C. to 10 ° C., more preferably −50 ° C. to −10 ° C., and particularly preferably −40 ° C. to −20 ° C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

Compound 2 can be produced by diesterizing oxalic acid by a known method, or a commercially available product may be used.

[Step 1-1]
In the reaction of step ¹ , compound 2-1 (a compound in which one of the hydroxy groups is protected by R6) or a mixture of compound 2-2 and compound 2-1 may be obtained. ^In that case, compound 2-2 can be obtained by deprotecting the silyl protecting group R6 of compound 2-1.
The reaction of step 1-1 can be carried out in the same manner as in step 1.

[Step 1-2]
Compound 2-2 can be obtained by deprotecting the silyl protecting group ^R6 of compound 2-1.
Deprotection can be performed in the presence of fluoride salts such as tetrabutylammonium fluoride (TBAF), cesium fluoride, hydrofluoride salts, or acids such as hydrochloric acid, acetic acid, paratoluenesulfonic acid.

The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include inert solvents such as tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, diethyl ether, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, and tetrahydrofuran is preferable. It is preferable to add acetic acid.

The amount of the fluoride salt is preferably 0.1 to 10 mol with respect to 1 mol of compound 2-1 (in the case of a mixture of compound 2-2 and compound 2-1). The amount of acid is preferably 0.1 to 10 mol with respect to 1 mol of compound 2-1 (1 mol of the mixture in the case of a mixture of compound 2-2 and compound 2-1). The reaction in step 1-2 is preferably carried out at a temperature of 50 ° C. or lower. By carrying out the reaction at a temperature of 50 ° C. or lower, compound 2-2 can be produced in high yield. The reaction temperature is preferably −80 ° C. to 50 ° C., more preferably −40 ° C. to 30 ° C., and particularly preferably −20 ° C. to 30 ° C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[Step 2]
Compound 3 can be obtained by subjecting compound 2-2 to a dehydration reaction.
The dehydration reaction can be carried out in the presence of a dehydrating agent such as diphosphorus pentoxide, concentrated sulfuric acid, calcium chloride, sodium sulfate, magnesium sulfate, calcium sulfate, molecular sieve (synthetic zeolite), and silica gel. As the dehydrating agent, diphosphorus pentoxide is preferable. The amount of the dehydrating agent is preferably 10 to 100% by weight based on 100% by weight of compound 2-2. The dehydration reaction can be carried out by distilling compound 2-2 in the presence of a dehydrating agent. Distillation is preferably carried out at a temperature of 30 ° C to 150 ° C. If the distillation temperature is too high, compound 3 may decompose. If the distillation temperature is too low, compound 3 cannot be condensed and the recovery rate may decrease. Distillation can be carried out at any pressure of reduced pressure, normal pressure and pressure, and can be appropriately determined so that the boiling point of compound 3 falls within the above-mentioned preferable temperature range. The pressure is preferably 0.1 mmHg to 5 atm (3800 mmHg).

[Step 3]
Compound 4 can be obtained by reacting compound 3 with compound 9 or compound 10.

In the general formula (9), R ² is a protecting group for an amino group as described above. R ⁷ , R ⁸ and R ⁹ are independently C _6-14 aryl groups. Examples of the C _6-14 aryl group represented by R ⁷ , R ⁸ or R ⁹ include a phenyl group and a naphthyl group. Preferably, R ⁷ , R ⁸ and R ⁹ are phenyl groups, respectively.

The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include inert solvents such as diethyl ether, tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, and diethyl ether is preferable.

The amount of compound 9 or compound 10 is preferably 0.5 to 10 mol with respect to 1 mol of compound 3. The reaction temperature is preferably −78 ° C. to 100 ° C., more preferably 0 ° C. to 40 ° C. The reaction time is preferably 1 minute to 24 hours, more preferably 10 minutes to 4 hours.

In a preferred embodiment, a carbamate-based protecting group such as a tert-butoxycarbonyl group or a 9-fluorenylmethyloxycarbonyl group is used as the amino group protecting group ^R2 to deprotect ^R2 under mild conditions. It is possible to synthesize fluorine-containing amino acids while suppressing the decomposition and racemization of compounds.

[Step 4]
Compound 5 can be obtained by subjecting compound 4 to a reduction reaction.
The reduction reaction can be carried out by a method using a reducing agent or a method of reducing in the presence of a metal catalyst.

(1) Method using a reducing agent As the reducing agent, sodium borohydride, zinc borohydride, sodium borohydride cyanoborohydride, lithium triethylborohydride, lithium borohydride (sec-butyl), lithium borohydride, and hydride tri. A boron borohydride reagent such as potassium boron (sec-butyl), lithium boron borohydride, and sodium triacetoxyborohydride can be used. As the reducing agent, sodium borohydride or zinc borohydride is preferable, and sodium borohydride is more preferable. The amount of the reducing agent is preferably 0.5 to 10 mol with respect to 1 mol of the compound 4.

The reaction can be carried out in a solvent inert to the reaction. Solvents include diethyl ether, dichloromethane, hydrochlorofluorocarbon (HCFC) (eg, Asahiclin® AK-225 (3,3-dichloro-1,1,1,2,2-pentafluoropropane and 1,1). 3-Dichloro-1,1,2,2,3-pentafluoropropane mixture, AGC Co., Ltd.)), dichloromethane, acetonitrile, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, etc. The inert solvent of the above is mentioned, and diethyl ether is preferable.
The reaction temperature is preferably −78 ° C. to 100 ° C., more preferably −10 ° C. to 40 ° C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

(2) Method of reduction in the presence of a metal catalyst Examples of the metal catalyst include palladium catalysts (eg, palladium carbon, palladium hydroxide, Pearlman catalyst, Lindler catalyst, silica gel-supported palladium catalyst, alumina-supported palladium catalyst, palladium oxide) and nickel. Catalysts (eg, lane nickel), platinum catalysts (eg, platinum carbon, platinum oxide, silica gel-supported platinum catalysts, alumina-supported platinum catalysts), rhodium catalysts (eg, rhodium carbon, alumina-supported rhodium catalysts, rhodium oxide), ruthenium catalysts (eg, rhodium oxide). , Luthenium carbon, alumina-supported ruthenium catalyst, ruthenium oxide), cobalt catalyst (eg, lane cobalt) and the like, and palladium catalyst is preferable. The amount of the metal catalyst is preferably 0.0001 to 0.1 mol, more preferably 0.0005 to 0.02 mol, based on 1 mol of the compound 4.

The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include inert solvents such as methanol, ethanol, isopropanol, diethyl ether, tetrahydrofuran, ethyl acetate, dichloromethane, acetonitrile, 1,4-dioxane, N, N-dimethylformamide and N, N-dimethylacetamide.

The reduction reaction is carried out in the presence of hydrogen gas. The reduction reaction may be carried out under normal pressure or under pressure. The pressure of hydrogen gas is preferably 0.5 atm to 10 atm. The reaction temperature is preferably 0 ° C to 100 ° C, more preferably 10 ° C to 50 ° C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[Step 5-1]
Compound 6-1 can be obtained by deprotecting the protecting group ^R2 of compound 5.
Deprotection can be performed according to the type of protecting group ^R2 .
When R ² is a Boc group, it can be deprotected under acidic conditions. Examples of the acid used include trifluoroacetic acid (TFA) and hydrochloric acid. The amount of acid is preferably 1 to 1000 mol with respect to 1 mol of compound 5.

The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include inert solvents such as diethyl ether, tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, and dichloromethane, N, N. -Dichloromethane amide is preferred. An acid can also be used as a solvent. Examples of the solvent include inorganic acids such as hydrochloric acid, acetic acid and trifluoroacetic acid, and organic acids, and trifluoroacetic acid is preferable. The reaction temperature is preferably −78 ° C. to 50 ° C., more preferably 0 ° C. to 40 ° C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

When R ² is an Fmoc group, it can be deprotected under basic conditions. Examples of the base used include secondary amines such as piperidine, morpholine and pyrrolidine. The amount of the base is preferably 1 to 100 mol with respect to 1 mol of the compound 5.
The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include inert solvents such as diethyl ether, tetrahydrofuran, dichloromethane, acetonitrile, benzene, toluene, 1,4-dioxane, N, N-dimethylformamide and N, N-dimethylacetamide. The reaction temperature is preferably −20 ° C. to 80 ° C., more preferably 0 ° C. to 40 ° C. The reaction time is preferably 1 minute to 24 hours, more preferably 5 minutes to 2 hours.

[Step 6-1]
Compound 7 can be obtained by deprotecting the protecting group ^R1 of compound 6-1.
Deprotection can be performed according to the type of protecting group ^R1 . When R ¹ is a benzyl group, a triphenylmethyl group, a 9-anthrylmethyl group, a piperonyl group, a 2- (9,10-dioxo) anthrylmethyl group, a benzyloxymethyl group or a phenacyl group, in the presence of a metal catalyst. It can be deprotected by the method of reducing with. The reduction reaction can be carried out in the same manner as the method of reduction in the presence of the metal catalyst in step 4.

[Step 5-2]
Compound 6-2 can be obtained by deprotecting the protecting group ^R1 of compound 5. Deprotection can be performed in the same manner as in step 6-1.

[Step 6-2]
Compound 7 can be obtained by deprotecting the protecting group ^R2 of compound 6-2. Deprotection can be performed in the same manner as in step 5-1.

By performing asymmetric reduction of imine (compound 4) represented by the general formula (4), an optically active fluorine-containing amino acid (fluoroalkyl group-containing compound) can be synthesized. In the following reaction formula, an asterisk (*) indicates that the absolute configuration of the asymmetric carbon atom with an asterisk is S or R. Further, Rf, R1 and R2 are as defined above.

In the production method, by using an aralkyl protecting group such as a benzyl group or a triphenylmethyl group as the protecting group ^R1 of the carboxy group, ^R1 can be deprotected under mild conditions and the optical activity is maintained. It is advantageous in that it is possible to synthesize a fluorine-containing amino acid and a fluorine-containing peptide.

[Step 7]
Compound 5-1 can be obtained by subjecting compound 4 to an asymmetric reduction reaction.
The asymmetric reduction reaction can be carried out by reducing compound 4 in the presence of an asymmetric reduction catalyst.

As the asymmetric reduction catalyst, a transition metal complex in which an asymmetric ligand is coordinated to the transition metal can be used. Examples of the transition metal include palladium, rhodium, ruthenium, iridium, nickel, cobalt, platinum, iron and the like. Examples of the transition metal complex include a palladium complex, a rhodium complex, a ruthenium complex, an iridium complex, a nickel complex and the like.

Asymmetric ligands include dpen (1,2-diphenylethylenediamine), daipen (1,1-di (4-anicil) -2-isopropyl-1,2-ethylenediamine), and optically active phosphine ligands. Can be mentioned. Optically active phosphinid ligands include 2,2'-bis (diphenylphosphino) -1,1'-binaphthyl (BINAP), 2,2'-bis (diphenylphosphino) -5,5', 6 , 6', 7,7', 8,8'-octahydro-1,1'-binaphthyl (H8-BINAP), 2,2'-bis (di-p-tolylphosphino) -1,1'-binaphthyl (Tol) -BINAP), 2,2'-bis [bis (3,5-dimethylphenyl) phosphino] -1,1'-binaphthyl (Xyl-BINAP), 2,2'-bis [bis (3,5-di-) tert-butyl-4-methoxyphenyl) phosphino] -1,1'-binaphthyl (DTBM-BINAP), 1,2-bis (anisylphosphino) ethane (DIPAMP), 2,3-bis (diphenylphosphino) Butane (CHIRAPHOS), 1-cyclohexyl-1,2-bis (diphenylphosphino) ethane (CYCPHOS), 1,2-bis (diphenylphosphino) propane (PROPHOS), 2,3-bis (diphenylphosphino)- 5-Norbornene (NORPHOS), 2,3-O-isopropylidene-2,3-dihydroxy-1,4-bis (diphenylphosphino) butane (DIOP), 1- [1', 2-bis (diphenylphosphino) ) Ferrosenyl] ethylamine (BPPFA), 1- [1', 2-bis (diphenylphosphino) ferrosenyl] ethyl alcohol (BPPFOH), 2,4-bis- (diphenylphosphino) pentane (SKEWPHOS), 1,2- Bis (substituted phosphorano) benzene (DuPHOS), 5,5'-bis (diphenylphosphino) -4,4'-bi-1,3-benzodioxol (SEGPHOS), 5,5'-bis [di (di (di (diphenylphosphino)) 3,5-kisilyl) phosphino] -4,4'-bi-1,3-benzodioxol (DM-SEGPHOS), 5,5'-bis [bis (3,5-di-tert-butyl-4) - -[2- (2'-2-substituted phosphinophenyl) ferrosenyl] ethyl-2-substituted phosphin (Walphos) and the like can be mentioned.

The amount of the asymmetric reduction catalyst is preferably 0.0001 to 0.1 mol, more preferably 0.0005 to 0.02 mol, based on 1 mol of the compound 4.
The reaction can be carried out in a solvent inert to the reaction. Examples of the solvent include inert solvents such as methanol, ethanol, isopropanol, diethyl ether, tetrahydrofuran, ethyl acetate, dichloromethane, acetonitrile, 1,4-dioxane, N, N-dimethylformamide and N, N-dimethylacetamide.
The reduction reaction is carried out in the presence of hydrogen gas. The reduction reaction may be carried out under normal pressure or under pressure. The pressure of hydrogen gas is preferably 0.5 atm to 10 atm. The reaction temperature is preferably 0 ° C to 100 ° C, more preferably 10 ° C to 50 ° C. The reaction time is preferably 1 to 48 hours, more preferably 6 to 36 hours.

[Step 8-1]
Compound 6-3 can be obtained by deprotecting the protecting group R2 of compound 5-1. Deprotection can be performed in the same manner as in step 5-1.

[Step 9-1]
Compound 7-1 can be obtained by deprotecting the protecting group R1 of compound 6-3. Deprotection can be performed in the same manner as in step 6-1.

[Step 8-2]
Compound 6-4 can be obtained by deprotecting the protecting group R1 of compound 5-1. Deprotection can be performed in the same manner as in step 6-1.

[Step 9-2]
Compound 7-1 can be obtained by deprotecting the protecting group R2 of compound 6-4. Deprotection can be performed in the same manner as in step 5-1.

The synthesis of optically active fluorine-containing amino acids (fluoroalkyl group-containing compounds) can also be carried out by the following reaction. In the following reaction formula, the asterisk indicates that the absolute configuration of the asymmetric carbon atom with the asterisk is S or R. Further, Rf, R ¹ and R ² are as defined above.

[Step 10-1]
Compound 6-3 can be obtained by optically resolving compound 6-1.
Optical resolution can be performed by a known method. For example, it can be performed by a method using a chiral column, a method by crystallization, a diastereomer method, or the like.

(1) Method using a chiral column A racemate can be divided into optically active substances by liquid chromatography or supercritical fluid chromatography (SFC) using a chiral column. As the chiral column, CHIRALPAK (registered trademark) (Daicel Co., Ltd.), CHIRALCEL (registered trademark) (Daicel Co., Ltd.) and the like can be used.

(2) Method by crystallization A salt of a racemate and an optically active amine or an optically active acid is formed and induced into a crystalline diasteremer salt for fractional crystallization. By repeating recrystallization, a single diastereomer salt can be obtained. If necessary, the diastereomeric salt is neutralized to obtain a free optically active substance. Examples of optically active amines include brucine, cinchonidine, cinchonine, 1-phenethylamine and the like. Examples of the optically active acid include camphorsulfonic acid, tartaric acid, mandelic acid and the like.

(3) Diastereomer method A racemic mixture is reacted with an optically active reagent to obtain a mixture of diastereomers, which is separated by fractional crystallization and chromatography to separate a single diastereomer. The optically active reagent moiety is removed from the obtained single diastereomer to obtain the desired optical isomer.

[Step 11-1]
Compound 7-1 can be obtained by deprotecting the protecting group R1 of compound 6-3. Deprotection can be performed in the same manner as in step 6-1.

[Step 10-2]
Compound 6-4 can be obtained by optically resolving compound 6-2. The optical resolution can be performed by the same method as in step 10-1.

[Step 11-2]
Compound 7-1 can be obtained by deprotecting the protecting group ^R2 of compound 6-4. Deprotection can be performed in the same manner as in step 5-1.

[Step 12]
Compound 7-1 can be obtained by optically resolving compound 7. The optical resolution can be performed by the same method as in step 10-1.

<Method for producing fluoroalkyl group-containing peptide>
The fluoroalkyl group-containing peptide can be produced from an amino acid having a fluoroalkyl group introduced in the side chain as a raw material. For example, a fluoroalkyl group-containing peptide can be produced by using compound 6-1 as a raw material, compound 6-2, compound 6-3, or compound 6-4 as a raw material.

For example, compound 6-2 or 6-4 is condensed with a carboxy group-protected amino acid, a carboxy group-protected amino acid, a C-terminal protected fluorinated peptide, or a C-terminal protected peptide. By allowing the peptide to be produced, a fluoroalkyl group-containing peptide can be produced. Further, the compound 6-1 or the compound 6-3 may be a fluorine-containing amino acid having an amino group protected, an amino acid having an amino group protected, a fluorine-containing peptide having an N-terminal protection, or a peptide having an N-terminal protection. By condensing, a fluoroalkyl group-containing peptide can be produced.

Further, after protecting the amino group or carboxy group of compound 7 or compound 7-1, a fluoroalkyl group-containing peptide can be produced in the same manner. Specifically, after protecting the amino group with a protective group, a carboxy group-protected amino acid containing a fluorine, an amino acid having a carboxy group protected, a C-terminal protected fluorine-containing peptide, or a C-terminal protected. Condensate with peptide. In addition, after protecting the carboxy group with a protective group, it is condensed with a fluorine-containing amino acid having an amino group protected, an amino acid having an amino group protected, a fluorine-containing peptide having an N-terminal protected, or a peptide having an N-terminal protected. You can also let it.

Fluoroalkyl groups have a high affinity for cell membranes. Therefore, the fluoroalkyl group-containing peptide according to the present invention has excellent cell membrane permeability. Moreover, since the structure is significantly different from that of the natural peptide, it is not easily decomposed by peptidase. Utilizing these properties, the fluoroalkyl group-containing peptide according to the present invention is expected to be used in the pharmaceutical field as a physiologically active substance. For example, the fluoroalkyl group-containing peptide according to the present invention can be expected to be used as a DDS carrier that carries a medicinal ingredient to a target cell. For example, by adding a fluoroalkyl group-containing peptide according to the present invention to a functional peptide that exhibits some physiological activity by being taken up into a target cell in the living body so as not to impair its function, the functionality thereof. The efficiency of peptide uptake into target cells can be improved. Further, a side chain of a part of the hydrophobic amino acid residues of the functional peptide exhibiting physiological activity is Rf, preferably the general formula (101) or (102), as long as the function of the functional peptide is not impaired. By substituting with the group represented by), the cell membrane permeability of the functional peptide and the residence time in the cell can be improved.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to these Examples.

The NMR apparatus used for the analysis of Examples and Comparative Examples was JEM-ECZ400S (400 MHz) manufactured by JEOL Ltd., and Tetramethylsilane was used as a reference value of 0PPM in ¹ H NMR and C ₆ F ₆ was used as a reference value of -162 PPM in ¹⁹ F NMR. did.

The following abbreviations are used herein.
Bn: BenzylBoc: t-butoxycarbonyl All: Allyl Phth: Phthalroyl Et ₂ O: Diethyl ether Fmoc: 9-Fluorenylmethyloxycarbonyl THF: Tetrahydrofuran TMS: Trimethylsilyl C ₄ F ₉ : 1,1,2,2 3,3,4,4,4-Nonafluorobutyl C ₆ F ₁₃ : 1,1,2,2,3,3,4,4,5,6,6,6-tridecafluorohexyl C ₈ F ₁₇ : 1,1,2,2,3,3,4,5,5,6,6,7,7,8,8,8-Heptadecafluorooctyl COMU: 1-cyano-2-ethoxy -2-oxoethylideneaminooxy) dimethylaminomorpholinocarbenium hexafluorophosphate (CAS RN: 1075198-30-9)
oxyma: (Hydroxyimino) Ethyl cyanoacetate (CAS RN: 3849-21-6)
DIPEA: Diisopropylethylamine

[Manufacturing Example 1]
2-((T-butoxycarbonyl) amino) -3,3,4,4,5,5,6,6-nonafluorocaproic acid was synthesized from trimethyl (nonafluorobutyl) silane and dibenzyl oxalate. ..

[Step 1]

Place the stir bar in an oven-dried 100 mL two-necked flask and add dibenzyl oxalate (5.41 g, 20.0 mmol), cesium fluoride (255 mg, 1.68 mmol), and THF (54 mL) under a nitrogen atmosphere. The mixture was further stirred, cooled to −30 ° C., trimethyl (nonafluorobutyl) silane (4.50 mL, 20.2 mmol) was added, and stirring was continued at −30 ° C. for 24 hours. A saturated aqueous ammonium chloride solution (30 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic phases are dried over sodium sulfate, filtered off, and the filtrate is distilled off under reduced pressure to 2- (benzyloxy) -3,3,4,4,5,5,6,6-nonafluoro-2. -A mixed crude product of benzyl ((trimethylsilyl) oxy) caproic acid and benzyl 3,3,4,4,5,5,6,6-nonafluoro-2,2-dihydroxycaproate was obtained. The obtained crude product was used in the next step without purification.

[Step 1-2]

Put the stir bar in a 100 mL two-necked flask dried in an oven, and under a nitrogen atmosphere, the total amount of the crude material obtained in step 1 and tetrabutylammonium fluoride (TBAF) 1 mol / L THF solution (10.5 mL, 10. 5 mmol), acetic acid (1 mL), and THF (50 mL) were added and stirred at 0 ° C. Then, the temperature was raised to room temperature, and after stirring for 24 hours, saturated aqueous sodium hydrogen carbonate (30 mL) was added for quenching, and the mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic phase was washed with water (50 mL) and saturated brine (50 mL), dried over sodium sulfate, filtered off, and the filtrate was distilled off under reduced pressure to 3,3,4,4,5,5,6. , 6,6-Nonafluoro-2,2-Dihydroxyhexanoate benzyl crude was obtained. The obtained crude product was used in the next step without purification.

^{1 1} H NMR (400 MHz, CDCl ₃ ) δ7.39 (brs, 5H), 5.37 (s, 2H).
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ-80.79 (brs, 3F), -121.09 (brs, 2F), -121.24-121.26 (m, 2F), -126.12-126 .21 (m, 2F).

[Step 2]

The stirrer was placed in a 20 mL flask dried in an oven, the total amount of the crude material obtained in step 2 and phosphorus pentoxide (22% by weight of the crude material) were added under a nitrogen atmosphere, and distillation was performed under reduced pressure. Fractions obtained at 2 mmHg, 77 ° C. were collected to give benzyl 3,3,4,4,5,5,6,6,6-nonafluoro-2-oxohexanoate as a colorless liquid (from step 1). Yield 73% through step 3).

^{1 1} H NMR (400 MHz, CDCl ₃ ) δ7.41 (brs, 5H), 5.40 (s, 2H).
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ-80.79 (brs, 3F), 117.78-117.850 (t, 2F, JF-F = 13 Hz), -122.01 (brs, 2F), -125.58 (brs, 2F).

Benzyl 3,3,4,4,5,5,6,6-nonafluoro-2-oxohexanoate was added in the same manner as in Steps 1 to 2 except that the temperature in Step 1 was changed to 0 ° C. Obtained as a colorless liquid. The yield from step 1 to step 2 was 69%.

[Step 3]

Place the stir bar in a 30 mL Schlenk flask dried in an oven and place it in a nitrogen atmosphere under a nitrogen atmosphere. 6 mmol), t-butyl (triphenylphosphaneilidene) carbamate (2.6 mmol), and Et2O (10 mL) were added. The reaction mixture was stirred at room temperature for 1 hour, filtered, and the filtrate was washed with Et2O (2 × 2 mL). The combined organic phases were distilled off under reduced pressure to obtain a crude product of 2-((t-butoxycarbonyl) imino) -3,3,4,4,5,5,6,6,6-benzyl nonafluorohexanoate. rice field. The obtained crude product was purified by silica gel chromatography (Et ₂ O / hexane = 1/4) and 2-((t-butoxycarbonyl) imino) -3,3,4,4,5,5,6. Benzyl 6,6-nonafluorohexaneate was obtained as a colorless liquid (yield 87%).

^{1 1} H NMR (400 MHz, CDCl ₃ ) δ7.410-7.352 (m, 5H), 5.350 (s, 2H), 1.504 (s, 9H).
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ-80.76-80.78 (t, 3F, JF-F = 9 Hz), -112.37 (brs, 2F), -121.0 (brs, 2F), -125.36 (brs, 2F).

[Step 4]

Put the stir bar in a 30 mL Schlenk flask dried in an oven, and under a nitrogen atmosphere, 2-((t-butoxycarbonyl) imino) -3,3,4,4,5,5,6,6-nona Benzyl fluorohexanoate (0.2 g, 0.42 mmol) was dissolved in Et2O (15 mL) and stirred at 0 ° C. Sodium borohydride (0.46 mmol) was added in 3 portions at 0 ° C., then the temperature was raised to room temperature and the mixture was stirred for 24 hours. Ice water was added for quenching, and 1 mol / L hydrochloric acid was added to bring the pH to less than 7. The aqueous phase was extracted with Et2O (2 × 10 mL), and the combined organic phase was distilled off under reduced pressure to 2-((t-butoxycarbonyl) amino) -3,3,4,4,5,5,6,6. , A crude material of benzyl 6-nonafluorocaproate was obtained. The obtained crude product was purified by silica gel chromatography (ethyl acetate / hexane = 1/4) and 2-((t-butoxycarbonyl) amino) -3,3,4,4,5,5,6,6. , Benzyl 6-Nonafluorohexaneate was obtained as a colorless liquid (yield 61%).

^{1 1} H NMR (400 MHz, CDCl ₃ ) δ7.40-7.33 (m, 5H), 5.41-5.39 (d, 2H, JH-H = 10 Hz), 5.28-5.20 (m) , 3H), 1.45 (s, 9H).
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ-80.86-80.89 (t, 3F, JF-F = 9 Hz), 115.36-118.55 (m, 2F), -121.50-123 .17 (m, 2F), -125.00-126.77 (m, 2F).

The same reaction as in Step 4 was carried out using the solvent and reducing agent (equivalent) shown in Table 1 instead of Et ₂ O and sodium borohydride. The yields are shown in Table 1. In the table, "AK225" refers to "Asahiclean® AK-225" (3,3-dichloro-1,1,1,2,2-pentafluoropropane and 1,3-dichloro-1,1,1. Amixture of 2,2,3-pentafluoropropane, AGC Co., Ltd.).

[Step 5-2]

Place the stirrer in an oven-dried 25 mL two-necked flask and place 2-((t-butoxycarbonyl) amino) -3,3,4,4,5,5,6,6-nonafluorohexanoic acid. Add benzyl (73.6 mg, 0.15 mmol), palladium / carbon (Pd 5%, about 55% water wet product, 20 mg), ethyl acetate (1 mL), and ethanol (7 mL), and add room temperature under normal pressure hydrogen atmosphere. Was stirred with. After stirring at room temperature for 24 hours, cerite filtration is performed, the filtrate is washed with ethanol (3 × 5 mL), and the combined organic phase is distilled off under reduced pressure to 2-((t-butoxycarbonyl) amino) -3,3. A crude product of 4,4,5,5,6,6,6-nonafluorohexanoic acid was obtained. The obtained crude product was purified by silica gel chromatography (ethyl acetate / hexane = 1/1) and 2-((t-butoxycarbonyl) amino) -3,3,4,4,5,5,6,6. , 6-Nonafluorohexaneic acid was obtained as a colorless liquid (yield 86%).

^{1 1} H NMR (400 MHz, CDCl ₃ ) δ7.72 (br, 1H), 5.49-5.47 (d, 2H, JH-H = 10 Hz), 5.24-5.15 (m, 1H), 1.45 (s, 9H).
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ-80.30 (brs, 3F), 114.64-118.03 (m, 2F), -120.70-122.40 (m, 2F), -124 .52-126.22 (m, 2F).

[Step 5-1]

The stir bar was placed in a 25 mL two-necked flask dried in an oven, and Boc-RFAA-OBn (376 mg, 0.78 mmol), 4M HCl, and 1,4-dioxane solution (3 mL) were added at 0 ° C. After stirring at room temperature for 18 hours, a saturated aqueous sodium carbonate solution was added to adjust the pH to be higher than 7, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over sodium sulfate. The organic phase was filtered and the filtrate was distilled off under reduced pressure to obtain a hydrochloride of H-RFAA-OBn as a white solid (yield 78%).

^{1 1} H NMR (400MHz, D2O) δ7.31 (brs, 2H), 6.81-6.77 (m, 5H), 5.27 (m, 1H), 3.71-3.67 (m, 2H) ).
¹⁹ F NMR (376 MHz, D2O) δ-80.38 (t, 3F), 118.29-121.00 (m, 2F), -121.00-123.80 (m, 2F), -126. 12-128.12 (m, 2F).

From now on, amino acids may be written in three-letter notation. For example, "Phe" is phenylalanine and "Gly" is glycine. Further, the peptide is described as (N-side protecting group) -amino acid three-letter notation- (C-side protecting group). "H-AA-OMe" means that the N-terminal side is unprotected and the C-terminal side is a methyl ester. When the C-terminal side is unprotected, it is expressed as "OH" instead of "OMe".

[Example 1]
A dipeptide having a nonafluorobutyl group was synthesized.

Place the stirrer in an oven-dried 25 mL two-necked flask and place 2-((t-butoxycarbonyl) amino) -3,3,4,4,5,5,6,6-nonafluorohexanoic acid. (33.4 mg, 0.085 mmol), DIPEA (0.13 mmol), DCM (3 mL), L-phenylalanine methyl ester (0.13 mmol), and benzotriazole-1-ol monohydrate (0.085 mmol). Was added at room temperature, cooled to 0 ° C., and then BOP (0.085 mmol) was added. After stirring at room temperature for 24 hours, the solvent was distilled off under reduced pressure, diluted with ethyl acetate, the organic phase was washed with saturated aqueous citrate solution, saturated aqueous sodium carbonate solution, and saturated aqueous sodium carbonate solution, and dried over sodium sulfate. The organic phase was filtered and the filtrate was distilled off under reduced pressure to obtain a crude product of Boc-RFAA (C4) -Phe-OMe. The obtained crude product was purified by silica gel chromatography (ethyl acetate / hexane = 1/4) to obtain two types of Boc-RFAA (C4) -Phe-OMe diastereomers as colorless liquids (two types). 22% yield including diastereomers).

Diastereomer A
^{1 1} H NMR (400 MHz, CDCl ₃ ) δ7.31-7.25 (m, 5H), 6.41-6.40 (d, NH, JH-H = 7 Hz), 5.48-5.46 (D, 1H, JH-H = 9Hz), 4.99-4.91 (m, 1H), 4.91-4.86 (m, 1H), 3.75 (s, 3H), 3.23 -3.10 (m, 2H), 1.47 (s, 9H).
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ-80.98 (t, 3F, JF-F = 7 Hz), 114.56-119.80 (m, 2F), -121.40-1323.22 (m) , 2F), -125.03-127.15 (m, 2F).

Diastereomer B
^{1 1} H NMR (400 MHz, CDCl ₃ ) δ7.30-7.07 (m, 5H), 6.42-6.40 (d, NH, JH-H = 9 Hz), 5.51-5.54 (D, 1H, JH-H = 8Hz), 5.00-4.95 (m, 1H), 4.93-4.88 (m, 1H), 3.74 (s, 3H), 3.15 -3.13 (m, 2H), 1.44 (s, 9H).
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ-80.77 (t, 3F, JF-F = 9 Hz), 113.74-119.30 (m, 2F), -121.22-122.94 (m) , 2F), -124.82-127.05 (m, 2F).

Here, using the crystal of diastereomer B, the solid of RFAA (C4) contained by X-ray structural analysis was determined to be the (S) form. Rigaku's VariMax Dual Saturn was used for the X-ray structure analysis.

The same reaction was carried out using the amino acid methyl esters shown in Table 2 instead of the L-phenylalanine methyl ester. The yields are shown in Table 2. In the table, "DCM" is dichloromethane, "BOP" is benzotriazole-1-yloxy-trisdimethylaminophosphonium hexafluorophosphate, and "EDC" is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. Represents.

[Example 2]
The protecting group on the N-terminal side of the peptide synthesized in Example 1 was deprotected.

Place the stir bar in an oven-dried 25 mL two-necked flask and place Boc-RFAA (C4) -Gly-OMe (21.2 mg, 0.05 mmol), DCM (1.5 mL), and TFA (0.4 mL). Was added. After stirring at room temperature for 24 hours, saturated aqueous sodium carbonate solution was added to adjust the pH to be higher than 7, and the mixture was extracted with DCM. The organic phase was washed with saturated brine and dried over sodium sulfate. The organic phase was filtered, and the filtrate was distilled off under reduced pressure to obtain a crude product of H-RFAA-Gly-OMe (yield 66%).

^{1 1} H NMR (400 MHz, CDCl ₃ ) δ7.08 (brs, 1H), 4.15-4.07 (m, 2H), 3.79 (s, 3H).
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ-80.71 (t, 3F, JF-F = 7 Hz), -115.13-119.94 (m, 2F), 119.94-121.93 (m) , 2F), -125.02-1269.82 (m, 2F).

[Example 3]
A tripeptide having a nonafluorobutyl group (Fmoc-Gly-RFAA (C4) -Gly-OMe) was synthesized.

H-RFAA (C4) -Gly-OMe (10.9 mg, 0.03 mmol), DCM (0.5 mL), DIPEA (0.13 mmol), Fmoc-Gly-OH (0) in an oven-dried NMR test tube. .03 mmol) and benzotriazole-1-yloxy-trisdimethylaminophosphonium salt (0.085 mmol) were added at room temperature. After allowing to stand at room temperature for 24 hours, the solvent was distilled off under reduced pressure, diluted with ethyl acetate, the organic phase was washed with saturated aqueous citrate solution, saturated aqueous sodium carbonate solution, and saturated aqueous sodium carbonate solution, and dried over sodium sulfate. The organic phase was filtered and the filtrate was distilled off under reduced pressure to obtain a crude product of Fmoc-Gly-RFAA-Gly-OMe.

^{1 1} H NMR (400 MHz, CDCl ₃ ) δ7.78-7.27 (m, 8H), δ7.30 (brs, 1H), 7.13 (brs, 1H), 5.63-5.61 (d, 1H, JH-H = 7Hz), 5.64-5.54 (m, 1H), 4.42-4.41 (d, 2H, JH-H = 7Hz), 4.24-4.20 (m) , 1H), 4.08-3.98 (m, 4H), 3.74 (s, 3H).
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ-80.78 (t, 3F, JF-F = 10 Hz), -114.61-118.74 (m, 2F), -121.22-123.09 (m) , 2F), -124.89-126.90 (m, 2F).

[Example 4]
A tripeptide having a nonafluorobutyl group (Boc-Ala-[(R) -RFAA (C4)]-Phe-OMe) was synthesized.

Place the stir bar in a 25 mL two-necked flask, and add Boc-[(R) -RFAA (C4)]-Phe-OMe (diastereomer A, DR> 95, 0.11 mmol) and DCM (5 mL) to one side. added. After cooling the reaction mixture to 0 ° C., TFA (1.25 mL) was added and the mixture was warmed to room temperature. After stirring for 4 hours, an aqueous sodium hydrogen carbonate solution was added to terminate the reaction. The aqueous phase was extracted with DCM, the combined organic phase was distilled off under reduced pressure, purified by silica gel column chromatography (hexane: ethyl acetate: triethylamine = 2: 1: 1%), and H-[(R)-. RFAA (C4)]-Phe-OMe was obtained (33.9 mg, yield 70.0%).

^{1 1} H NMR (400 MHz, CDCl ₃ ) δ = 7.27 (m, 5H), 6.81-6.83 (d, NH), 4.90-4.95 (dd, 1H), 3.93- 3.99 (dd, 1H), 3.74 (s, 3H), 3.10-3.14 (m, 2H), 1.77 (br s, 2H)
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ = -80.7 (t, 3F), -120.5-114.4 (m, 2F), -121.2-119.7 (m, 2F),- 126.2-125.7 (m, 2F)

Place the stir bar in a 25 mL two-necked flask, while adding Boc-Ala-OH (1.1 equal volume) and 1-hydroxy-7-azabenzotriazole (HOAt, 1.1 equal volume) to 3 mL DCM. ), DIPEA (1.3 equal amount), 1- [Bis (dimethylamino) methylene] -1H-1,2,3-triazole [4,5-b] pyridinium 3-Oxide Hexafluorophosphate (HATU, 1.1 equal amount) ) And a dipeptide (H-[(R) -RFAA (C4)]-Phe-OMe, dr> 95: 5,38 μmol) were added to bring the temperature to 0 ° C. The mixture was warmed to room temperature and then stirred for 1.5 hours. Then, HCl (1N) was added to terminate the reaction. The reaction mixture was separated by HCl (1N) and DCM, the combined organic phase was distilled off under reduced pressure, and then the mixture was diluted with ethyl acetate. The organic phase was washed with HCl (1N), saturated aqueous NaHCO ₃ solution, and brine, dried over Na ₂ SO ₄ and evaporated to give a white solid. The crude mixture was purified by silica gel column chromatography (hexane: ethyl acetate = 2: 1) to give a tripeptide (dr> 95: 5, 18.2 mg, yield 75.1%).

^{1 1} H NMR (400 MHz, Deuterated d6) δ = 8.30 (d, NH), 7.82 (d, NH), 7.19-7.29 (m, 5H), 6.23 ( d, NH), 5.53-5.61 (m, 1H), 4.68-4.77 (m, 1H), 4.26-4.29 (m, 1H), 3.66 ( s, 3H), 3.04-3.16 (m, 2H), 1.38 (s, 9H), 1.30 (d, 3H)
¹⁹ F NMR (376 MHz, Deuterated d6) δ = -81.5 (t, 3F), -120.1-115.0 (m, 2F),-123.1-121.9 (m, 2F),- 126.9-126.3 (m, 2F)

[Example 5]
A tripeptide having a nonafluorobutyl group (H-Ala-[(R) -RFAA (C4)]-Phe-OMe) was synthesized.

A stir bar was placed in a 25 mL two-necked flask, and Boc-[(R) -RFAA (C4)]-Phe-OMe (diastereomer A, DR> 95,29 μmol) and DCM (2 mL) were added to one of them. .. After cooling the reaction mixture to 0 ° C., TFA (0.4 mL) was added and the mixture was warmed to room temperature. After stirring for 4 hours, an aqueous sodium hydrogen carbonate solution was added to terminate the reaction. The aqueous phase was extracted with DCM, the combined organic phase was distilled off under reduced pressure, purified by silica gel column chromatography (CHCl ₃ : MeOH = 10: 1), and H-Ala-[(R) -RFAA (C4). )]-Phe-OMe (dr> 95, 13.8 mg, yield 90.6%).

^{1 1} H NMR (400 MHz, Deuterated d6) δ = 8.33 (d, NH), 8.16 (d, NH), 7.19-7.27 (m, 5H), 5.49- 5.56 (m, 1H), 4.68-4.74 (m, 1H), 3.94-3.99 (m, 1H), 3.67 (s, 3H), 3.00-3. 14 (m, 2H), 2.81 (br s, 2H), 1.16 (d, 3H)
¹⁹ F NMR (376 MHz, Deuterated d6) δ = -81.7 (t, 3F), -120.0-115.3 (m, 2F),-123.1-122.1 (m, 2F),- 126.9-125.6 (m, 2F)

[Example 6]
The fluorescent substance Alexa Fluor 647 was fused to the N-terminal of the tripeptide having a nonafluorobutyl group (H-Ala-[(R) -RFAA (C4)]-Phe-OMe) synthesized in Example 5 (Alexa). -Ala-[(R) -RFAA (C4)]-Phe-OMe).

AlexaFluor 647 (250 μg) dissolved in dry DMSO (15 μL) and H-Ala-[(R) -RFAA (C4)]-Phe dissolved in dry DMSO (15 μL) in a 1.5 mL black tube. -OMe (1.5 eq) and DIPEA (1.5 eq) were added. The mixture was continuously stirred at room temperature overnight. The mixture is purified by reverse phase chromatography (acetonitrile / water / TFA = 30: 70: 0.1 to 95: 5: 0.1), freeze-dried and Alexa-Ala-[(R) -RFAA (C4). )]-Phe-OMe was obtained as a blue solid (yield 32.3% calculated with a fluorometer). The fluorescence was measured at an emission wavelength of 650 nm using a NanoDrop (registered trademark) spectrophotometer ND-1000.

MALDI-TOF MS
[M] ^- : m / z calcd. for C ₅₅ H ₆₃ F ₉ N ₅ O ₁₇ S _4-1364.2964 , found 1364.7252

[Comparative Example 1]
A dipeptide having a butyl group (H-Nle-Phe-OMe) was synthesized.

Boc-Nle-Phe-Ome was synthesized according to the previous report (Chemical and Pharmaceutical Bulletin, 1987, vol.35, p.468) (yield 556 mg, yield 40.2%).

A stir bar was placed in a 25 mL two-necked flask, and Boc-Nle-Phe-OMe (1.42 mmol) and DCM (10 mL) were added to one of them. After cooling the reaction mixture to 0 ° C., TFA (2 mL) was added and the mixture was warmed to room temperature. After stirring for 4 hours, an aqueous sodium hydrogen carbonate solution was added to terminate the reaction. The aqueous phase was extracted with DCM, and the combined organic phase was distilled off under reduced pressure to obtain a stoichiometric amount of H-Nle-Phe-OMe. The product was used without further purification.

^{1 1} H NMR (400 MHz, ACETONE-D6)
Conformater A
Δ8.09-7.88 (m, 1H), 7.42-7.04 (m, 5H), 4.67 (dd, J = 11.0, 4.6Hz, 1H), 4.59 (t) , J = 7.5Hz, 1H), 3.69 (s, 3H), 3.22 (dd, J = 13.7, 4.6Hz, 1H), 3.00 (dd, J = 14.0, 10.7Hz, 1H), 2.00-1.80 (m, 2H), 1.48-1.18 (m, 2H), 0.83-0.73 (m, 3H)
Conformater B
7.60 (t, J = 7.8Hz, 1H), 7.42-7.04 (m, 5H), 4.75 (t, J = 6.9Hz, 1H), 4.25 (t, J) = 6.4Hz, 1H), 3.64 (s, 3H), 3.10 (t, J = 7.3Hz, 2H), 2.00-1.80 (m, 2H), 1.48-1 .18 (m, 2H), 0.86 (t, J = 7.3Hz, 3H)

[Comparative Example 2]
A tripeptide having a butyl group (H-Ala-Nle-Phe-OMe) was synthesized.

Fmoc-Ala-OH (1.1 eq), HOAt (1.1 eq), and DIPEA (1.3 eq) were added to a 50 mL two-necked round bottom flask. HATU (1.1 eq) and a dipeptide (H-N1-Phe-OMe, 1.42 mmol) dissolved in 20 mL of DCM were added to the mixture at 0 ° C. The mixture was warmed to room temperature, then stirred for 1.5 hours, and then HCl (1N) was added to terminate the reaction. The mixture was partitioned between HCl (1N) and DCM. The combined organic phases were distilled off under reduced pressure and then diluted with ethyl acetate. The organic phase was washed with HCl (1N), saturated aqueous NaHCO ₃ solution, and brine, dried over Na ₂ SO ₄ and evaporated to give a white solid. To the crude mixture was added 25 mL of a 20% piperidine DMF solution and stirred at room temperature for 1 hour. The solvent was removed by vacuum drying to give a white solid, which was purified by silica gel column chromatography (Et2O: DCM = 1: 3) to give H-Ala-Nle-Phe-OMe (dr> 95, 116 mg, yield 23.0%).

MALDI-TOF MS
[M + H] ⁺ : m / z calcd. for 364.22, found 364.07
[M + H] ⁺ : m / z calcd. for 386.21, found 386.06

[Comparative Example 3]
The fluorescent substance Alexa Fluor 647 was fused to the N-terminal of the butyl group-containing tripeptide (H-Ala-Nle-Phe-OMe) synthesized in Comparative Example 2 (Alexa-Ala-Nle-Phe-OMe).

AlexaFluor 647 (250 μg) dissolved in dry DMSO (15 μL), H-Ala-Nle-Phe-OMe (1.5 eq) dissolved in dry DMSO (15 μL), in a 1.5 mL black tube. And DIPEA (1.5 eq) were added. The mixture was continuously stirred at room temperature overnight. The mixture is purified by reverse phase chromatography (acetonitrile / water / TFA = 30: 70: 0.1-95: 5: 0.1), lyophilized and made Alexa-Ala-Nle-Phe-OMe blue. Obtained as a solid (yield 74.5% calculated by a fluorometer, emission wavelength = 650 nm).

MALDI-TOF MS
[M] ^- : m / z calcd. for C ₅₅ H ₇₂ N ₅ O ₁₇ S _4-1202.3812 , found 1202.1449

[Manufacturing Example 2]
From heptadecafluorooctyliodide and dibenzyl oxalate, 2-((t-butoxycarbonyl) amino) -3,3,4,4,5,5,6,6,7,8,8,9, Benzyl 9,10,10,10-heptadecafluorododecanoate was synthesized.

The perfluoroalkylation reaction was carried out according to the previous report (Journal of Fluorine Chemistry, 1984, vol.26, p.341-358).
The obtained crude product was purified by sublimation (72 ° C., 0.5 mmHg). The obtained white solid was directly transferred to a 100 mL volumetric round-bottom flask, dissolved in Et2O (10 mL), and reacted with tert-butyl (triphenylphosphinelidene) carbamate (5.5 mmol) at room temperature for 1 hour. The crude product was filtered and the filtrate was evaporated. The obtained white solid was purified by silica gel column chromatography (hexane: ethyl acetate = 10: 1w / 0.4% NEt3) to obtain α-imino ester (311 mg, 3 step yield: 8.2%). ..

^{1 1} H NMR (400 MHz, CDCl3) δ = 7.36-7.38 (m, 5H), 5.36 (s, 2H), 1.50 (s, 9H)
¹⁹ F NMR (376 MHz, CDCl3) δ = -81.0 (t, 3F), -112.6 (m, 2F), -120.2 (m, 2F), -121.2 (m, 2F), -121.8-122.0 (m, 4F), -122.8 (m, 2F), -126.3 (m, 2F)

A stir bar was placed in a 25 mL two-necked round bottom flask, and α-imino ester (311 mg, 0.47 mmol) and THF (5 mL) were added. Sodium triacetoxyborohydride (0.59 mmol) was added to the reaction mixture at 0 ° C. and then stirred at room temperature for 24 hours. The reaction mixture was evaporated directly and partitioned between water and DCM. The combined organic phases were distilled off under reduced pressure to obtain a crude mixture. The crude mixture was purified by silica gel column chromatography (ethyl acetate / hexane = 1:10) to give a white solid (Boc-RFAA (C8) -OBn) (yield 49.6%).

^{1 1} H NMR (400 MHz CDCl ₃ ) δ = 7.18 (m, 5H), 5.137 (m, 1H), 5.07 (s, 2H), 1.28 (s, 9H)
¹⁹ F NMR (376MHz CDCl ₃ ) δ = -81.0 (t, 3F), -115.3118.8 (m, 2F), -121.5-122.1 (m, 8F), -122 .9 (m, 2F), -126.3 (m, 2F)

[Manufacturing Example 3]
From heptadecafluorooctyliodide and diallyl oxalate, 2-((t-butoxycarbonyl) amino) -3,3,4,4,5,5,6,6,7,8,8,9, 9,10,10,10-heptadecafluorododecanoic acid was synthesized.

3,3,4,4,5,5,6,6,7,7,8,8 in the same manner as in Production Example 2 except that diallyl oxalate (3.4 g) was used instead of dibenzyl oxalate. , 9,9,10,10,10-A crude product of allyl hexadecafluoro-2,2-dihydrodecanoate was obtained in a yield of 80.9%. The obtained product was used in the next step without purification.

^{1 1} H NMR (400MHz Deuterated-d6) δ = 6.25-5.71 (m, 1H), 5.65-5.03 (m, 2H), 4.97-4.52 (m, 2H)
¹⁹ F NMR (376MHz Deuterated-d6) δ = 81.72 (m, 3F), -120.30 (s, 4F), -122.24 (s, 6F), -123.26 (s, 2F), -126.79 (d, J = 54.5Hz, 2F)

The obtained crude product was sublimated and purified at 64 ° C. and 2.2 mmHg in the same manner as in Example 7, with 3,3,4,4,5,5,6,6,7,7,8, Allyl 8,9,9,10,10,10-hexadecafluoro-2-oxodecanoate was obtained in a yield of 60.3%.

^{1 1} H NMR (400 MHz, CDCl ₃ ) δ = 6.08-5.79 (m, 1H), 5.59-5.13 (m, 2H), 4.89-4.80 (m, 2H)
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ = -81.03 (t, J = 10.0 Hz, 3F), 117.88 (t, J = 12.9 Hz, 2F), -121.25 (m, 4F), -121.96 (m4F), -122.85 (s, 2F), -126.31 (d, J = 5.7Hz, 2F)

Next, in silica gel chromatography, the α-imino ester yield was 95 in the same manner as in Example 7 except that the eluent was prepared by adding 1% triethylamine to hexane: ethyl acetate = 9: 1. Obtained at 1%.

^{1 1} H NMR (400 MHz, CDCl ₃ ) δ = 6.01-5.82 (m, 1H), 5.49-5.30 (m, 2H), 4.81 (d, J = 5.9 Hz, 2H) ), 1.63-1.49 (m, 9H)
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ = -79.98 to -82.00 (m, 3F), -112.58 (m, 2F), -120.10 (s, 2F), -121.11 (S, 2F), -121.80 (m, 4F), -122.71 (s, 2F), -126.38 (m, 2F)

2-Picoline borane (1 equivalent) was added to a solution of the obtained imino ester (2.0 g, 3.2 mmol) in dry diethyl ether (30 mL) at 0 ° C. with stirring. The reaction mixture was stirred at room temperature for 1.5 hours and then diluted with HCl (1N) (20 mL). The organic phase was washed twice with HCl (1N) and concentrated under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (hexane: ethyl acetate = 9: 1) to obtain α-butoxycarbonylamino ester. Obtained as a yellow solid (yield 54%).

^{1 1} H NMR (400 MHz, CDCl ₃ ) δ6.01-5.79 (m, 1H), 5.51-4.97 (m, 4H), 4.84-4.62 (m, 2H), 1. 57-1.35 (m, 9H)
¹⁹ F NMR (376MHz, CDCl ₃ ) δ-80.96 (t, J = 10.0Hz, 3F), -115.81 (d, J = 280Hz, 1F), -118.10 (d, J = 281Hz) , 1F), -120.88 to -123.39 (m, 10F), -126.23 (m, 2F)

Phenylsilane (2 equivalents) and tetrakistriphenylphosphine palladium (5 mol%) were added to a solution of the obtained α-butoxycarbonylamino ester (1.2 g, 1.9 mmol) in THF (18 mL) at 0 ° C. to room temperature. Was stirred for 2 hours. The reaction mixture was diluted with HCl (1N) (10 mL) and extracted twice with DCM. The organic phase was concentrated under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (chloroform: methanol = 6: 1/1% acetic acid) to 2-((tert-butoxycarbonyl) amino) -3, 3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorododecanoic acid was obtained as a pale yellow liquid (yield 74%). ..

^{1 1} H NMR (400 MHz, CDCl ₃ ) δ = 5.03 (m, J = 8.4 Hz, 1H), 1.38 (s, 9H)
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ = -80.87 (t, J = 10.0 Hz, 3F), -115.78 (d, J = 281.1 Hz, 1F), -118.12 (d, J = 281.1Hz, 1F), -120.16 to -123.43 (m, 10F), -126.19 (s, 2F)

[Example 7]
A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Phe-OMe) was synthesized.

First, in the same manner as in Production Example 3, 2-((tert-butoxycarbonyl) amino) -3,3,4,4,5,5,6,6,7,7,8,8,9,9, 10,10,10-Heptadecafluorododecanoic acid was synthesized. Then, in a dried 300 mL three-necked flask, 2-((tert-butoxycarbonyl) amino) -3,3,4,4,5,5,6,6,7,7,8, 8,9,9,10,10,10-heptadecafluorododecanoic acid (994.4 mg, 1.68 mmol), DCM (99 mL), phenylalanine methyl ester hydrochloride (1.84 mmol), and oxyma (1.84 mmol). Was mixed and stirred. The reaction mixture was cooled to 0 ° C., COMU (1.84 mmol) and DIPEA (3.69 mmol) were added, and the mixture was stirred at room temperature for 20.5 hours. The reaction mixture was then quenched with HCl (1N) and extracted 3 times with DCM. The combined organic phases were concentrated under reduced pressure, diluted with ethyl acetate, and washed with HCl (1N), saturated aqueous sodium hydrogen carbonate, and saturated brine. The washed organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product of Boc-RFAA (C8) -Phe-OMe. The crude product was purified by silica gel chromatography (ethyl acetate / hexane = 1/4) and Boc-[(R) -RFAA (C8)]-Phe-OMe (diastereomer A, 383.3 mg, 0. 508 mmol) and Boc-[(S) -RFAA (C8)]-Phe-OMe (diastereomer B, 417.1 mg, 0.553 mmol) total 807.3 mg (1.07 mmol, yield 63.8%). ) Was obtained.

Here, using the crystal of diastereomer B, the solid of RFAA (C8) contained by X-ray structural analysis was determined to be the (S) form. Rigaku's VariMax Dual Saturn was used for the X-ray structure analysis.

Boc-[(R) -RFAA (C8)]-Phe-OMe (Diastereomer A)
^{1 1} H NMR (500 MHz, CDCl ₃ ) δ = 7.30-7.25 (m, 3H), 7.07-7.05 (m, 2H), 6.40 (d, 1H), 5.46 ( m, 1H), 5.00-4.87 (m, 2H), 3.77 (s, 3H), 3.20-3.11 (m, 2H), 1.47 (s, 9H)
¹⁹ F NMR (470MHz, CDCl ₃ ) δ = -80.86 (t, 3F), -114.89 (d, J = 281Hz, 1F), -119.11 (d, J = 279Hz, 1F),- 120.40 to -123.10 (m, 10F), -126.23 (m, 2F)
Boc-RFAA (C8) -Phe-OMe

Boc-[(S) -RFAA (C8)]-Phe-OMe (Diastereomer B) ^{1 1} H NMR (500MHz, CDCl ₃ ) δ = 7.28-7.26 (m, 3H), 7.10- 7.04 (m, 2H), 6.35 (d, 1H), 5.48 (m, 1H), 5.00-4.87 (m, 2H), 3.74 (s, 3H), 3 .15-3.13 (m, 2H), 1.47 (s, 9H)
¹⁹ F NMR (470 MHz, CDCl ₃ ) -80.86 (t, 3F), -114.21 (d, J = 275 Hz, 1F), -118.78 (d, J = 279 Hz, 1F), -120. 35 to -123.00 (m, 10F), -126.23 (m, 2F)

The protecting group on the N-terminal side of the synthesized peptide was deprotected to obtain H-RFAA (C8) -Phe-OMe.

A stir bar was placed in a 50 mL two-necked flask, and Boc-[(R) -RFAA (C8)]-Phe-OMe (diastereomer A, 0.508 mmol) and DCM (17 mL) were added. After cooling the reaction mixture to 0 ° C., TFA (3.4 mL) was added and the temperature was raised to room temperature. After stirring for 1.5 hours, an aqueous sodium hydrogen carbonate solution was added to terminate the reaction. The aqueous phase was extracted with DCM and the combined organic phase was distilled off under reduced pressure to obtain a crude product of the deprotected H-[(R) -RFAA (C8)]-Phe-OMe. The crude product was purified by silica gel chromatography (ethyl acetate / hexane = 2/5) to give H-[(R) -RFAA (C8)]-Phe-OMe (diastereomers A) (0. 393 mmol, yield 67.7%).

H-[(R) -RFAA (C8)]-Phe-OMe (Diastereomer A)
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 7.95 (br, 1H), 7.30-7.22 (m, 5H), 4.82-4.77 (m, 1H), 4.40 -4.25 (m, 1H), 3.67 (s, 3H), 3.20-3.07 (m, 2H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), -115.00 (d, J = 277Hz, 1F), -117.20 (d, J = 275Hz, 1F), -120.00 to -122.70 (m, 10F), -125.95 (s, 2F)

Boc-[(S) -RFAA (C8)]-Phe-OMe (diastereomer B, 98.7 μmol) was deprotected in the same manner, and H-[(S) -RFAA (C8)]- Phe-OMe (diastereomer B) was obtained (60.1 μmol, yield 60.8%).

H-[(S) -RFAA (C8)]-Phe-OMe (Diastereomer B)
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 7.31-7.19 (m, 5H), 4.82-4.79 (m, 1H), 4.70-4.64 (m, 1H) , 3.72 (s, 3H), 3.22-3.06 (m, 2H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), -114.76 (d, J = 267Hz, 1F), -117.31 (d, J = 275Hz, 1F), -120.00 to -122.70 (m, 10F), -125.94 (s, 2F)

[Example 8]
A tripeptide having a heptadecafluorooctyl group (Boc-Ala-RFAA (C8) -Phe-OMe) was synthesized.

Dipeptide (H-[(R) -RFAA (C8)]-Phe-OMe, diastereomer A) (0.24 mmol), Boc-Ala-OH (1.2 eq) in a 25 mL two-necked round bottom flask. ) And oxyma (1.2 eq) were dissolved in 3.4 mL of DCM and stirred. The reaction mixture was cooled to 0 ° C., COMU (1.2 eq) and DIPEA (1.2 eq) were added, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was then quenched with HCl (1N) and extracted 3 times with DCM. The combined organic phases were concentrated under reduced pressure, diluted with ethyl acetate, and washed with HCl (1N), saturated aqueous sodium hydrogen carbonate, and saturated brine. The washed organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product of Boc-Ala-[(R) -RFAA (C8)]-Phe-OMe. The crude product was dissolved in DCM, reprecipitated with hexane and filtered to give pure Boc-Ala-[(R) -RFAA (C8)]-Phe-OMe (0.20 mmol, yield). 85.4%).

Boc-Ala-[(R) -RFAA (C8)]-Phe-OMe (Diastereomer A)
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.29 (br, 1H), 7.72 (br, 1H), 7.28-7.17 (m, 5H), 6.38 (br, 1H) ), 5.67-5.60 (m, 1H), 4.80-4.76 (m, 1H), 4.23-4.18 (m, 1H), 3.67 (s, 3H), 3.20-3.00 (m, 2H), 1.43 (s, 9H) 1.30 (t, 3H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), -114.57 (d, J = 282Hz, 1F), 119.31 (d, J = 281Hz, 1F), -120.00 to -122.70 (m, 10F), -125.96 (m, 2F)

H-[(S) -RFAA (C8)]-Phe-OMe (diastereomers B, 46.1 μmol) was condensed with Boc-Ala-OH by the same method, and Boc-Ala-[(S). ) -RFAA (C8)]-Phe-OMe (diastereomer B) was obtained (45.7 μmol, 99% yield).

Boc-Ala-[(S) -RFAA (C8)]-Phe-OMe (Diastereomer B)
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.33 (br, 1H), 7.85 (br, 1H), 7.30-7.22 (m, 5H), 6.30 (br, 1H) ), 5.63-5.53 (m, 1H), 4.72-4.67 (m, 1H), 4.32-4.25 (m, 1H), 3.66 (s, 3H), 3.18-3.02 (m, 2H), 1.40 (s, 9H) 1.32 (d, 3H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), -114.50 (d, J = 287Hz, 1F), -118.77 (d, J = 277Hz, 1F), -120.75 to -122.53 (m, 10F), -125.93 (m, 2F)

The protecting group on the N-terminal side of the synthesized peptide was deprotected to obtain H-Ala-[(R) -RFAA (C8)]-Phe-OMe.

A stir bar was placed in a 20 mL two-necked flask, and Boc-Ala-[(R) -RFAA (C8)]-Phe-OMe (diastereomer A, 0.194 mmol) and DCM (5 mL) were added to one of them. .. After cooling the reaction mixture to 0 ° C., TFA (1 mL) was added, the temperature was raised to room temperature, and the mixture was stirred for 2 hours. Then, an aqueous sodium hydrogen carbonate solution was added to terminate the reaction. The aqueous phase of the reaction mixture was extracted with DCM and the combined organic phase was distilled off under reduced pressure to obtain a crude product of the deprotected H-[(R) -RFAA (C8)]-Phe-OMe. The crude product was purified by silica gel chromatography (chloroform / methanol = 15/1) to give H-Ala-[(R) -RFAA (C8)]-Phe-OMe (diastereomer A) (0). .147 mmol, yield 75.6%).

H-Ala-[(R) -RFAA (C8)]-Phe-OMe (Diastereomer A)
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.30, 8.20 (m, NH), 7.28-7.19 (m, 5H), 6.38 (br, 1H), 5.68 -5.53 (m, 1H), 4.79-4.76 (m, 1H), 3.97-3.47 (m, 1H), 3.67 (s, 3H), 1.26,1 .17 (s, 3H)
¹⁹ F NMR (470 MHz, ACETONE-D6) δ = -80.86 (t, 3F), -114.50 (d, J = 282 Hz, 1F), 119.48 (d, J = 281 Hz, 1F), -120.40 to -122.65 (m, 10F), -125.92 (m, 2F)

Boc-Ala-[(S) -RFAA (C8)]-Phe-OMe (diastereomer B, 45.7 μmol) was deprotected in the same manner, and H-Ala-[(S) -RFAA ( C8)]-Phe-OMe (diastereomer B) was obtained (21.1 μmol, yield 46.2%).

H-Ala-[(S) -RFAA (C8)]-Phe-OMe (Diastereomer B)
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.38, 8.21 (m, NH), 7.31-7.21 (m, 5H), 5.59-5.54 (m, 1H) , 4.76-4.72 (m, 1H), 4.06-3.97 (m, 1H), 3.67 (s, 3H), 3.20-3.04 (m, 2H), 1 .19, 1.17 (s, 3H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), -114.82 (d, J = 282Hz, 1F), 118.68 (d, J = 284Hz, 1F), -120.80 to -122.70 (m, 10F), -125.98 (m, 2F)

The fluorescent substance AlexaFluor 647 was bound to the N-terminal of the synthesized deprotected tripeptide (H-Ala-[(R) -RFAA (C8)]-Phe-OMe).

Alexa Fluor 647 NHS ester (1.75 mg) dissolved in dry DMSO (175 μL) and H-Ala-[(R) -RFAA (C8) dissolved in dry DMSO (50 μL) in a 1.5 mL black tube. )]-Phe-OMe (diastereomer A, 3.0 equivalents), DIPEA dissolved in dry DMSO (71 μL) (3.0 equivalents), and dried DMSO 124 μL were added. The mixture was continuously stirred at room temperature overnight. The mixture was purified by reverse phase chromatography (acetonitrile / water / TFA = 25: 75: 0.1 to 99: 1: 0.1) and lyophilized to give fluorescent conjugate 1 as a blue solid (fluorescence). Calculated in total, yield 52.0%). The fluorescence was measured at a emission wavelength of 650 nm using a NanoDrop (registered trademark) spectrophotometer ND-1000.

MALDI-TOF MS
[M + 2] ^- : m / z calcd for C ₅₉ H ₆₃ F ₁₇ N ₅ O ₁₇ S _4-1564.2825 , found1564.3501

H-Ala-[(S) -RFAA (C8)]-Phe-OMe (diastereomer B, 1.241 μmol) was bound to AlexaFluor 647 in the same manner, and H-Ala-[(S). ) -RFAA (C8)]-Phe-OMe (diastereomer B) fluorescence conjugate 2 was obtained as a blue solid (calculated with a fluorometer, yield 54.3%, dye standard).

MALDI-TOF MS
[M + 4] ⁺ : m / z calcd for C ₅₉ H ₆₄ F ₁₇ N ₅ O ₁₇ S _4-1566.3935 , found1566.3679

[Example 9]
A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Gly-OMe) was synthesized.

In a dried 25 mL two-necked flask, 2-((tert-butoxycarbonyl) amino) -3,3,4,4,5,5,6,6,7,7,8,8, 9,9,10,10,10-heptadecafluorododecanoic acid (15.4 mg, 26.0 μmol), DCM (2 mL), DIPEA (57 μmol), glycine methyl ester hydrochloride (29 μmol), and oxyma (29 μmol). , Mixed and stirred. The reaction mixture was cooled to 0 ° C., COMU (29 μmol) was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction mixture was then quenched with HCl (1N) and extracted 3 times with DCM. The combined organic phases were concentrated under reduced pressure, diluted with ethyl acetate, and washed with HCl (1N), saturated aqueous sodium hydrogen carbonate, and saturated brine. The washed organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product of Boc-RFAA (C8) -Gly-OMe. The crude product was purified by silica gel chromatography (ethyl acetate / hexane = 1/3) to give Boc-RFAA (C8) -Gly-OMe. (Yield 92%)

^{1 1} H NMR (400 MHz, Deuterated-d6) δ = 8.25 (t, J = 5.3 Hz, 1H), 6.66 (d, J = 9.6 Hz, 1H), 5.44-5.19 ( m, 1H), 4.07 (d, J = 5.5Hz, 2H), 3.68 (s, 3H), 1.42 (s, 9H)
¹⁹ F NMR (376 MHz, Deuterated-d6) δ = -81.53 (s, 3F), -115.31 (d, J = 281.1 Hz, 1F), 119.15 to -120.92 (m, 1F), -120.92 to -124.38 (10F), 125.54 to -127.82 (m, 2F)

[Example 10]
A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Ala-OMe) was synthesized.

2-((tert-butoxycarbonyl) amino) -3,3,4,5,5,5 in the same manner as in Example 9 except that alanine methyl ester hydrochloride was used instead of glycine methyl ester hydrochloride. From 6,6,7,7,8,8,9,9,10,10,10-heptadecafluorododecanoic acid (15 μmol) to Boc-RFAA (C8) -Ala-OMe (diasteromer A and diastereomer) A 47:53 mixture of B) was obtained (yield 74%).

^{1 1} H NMR (400 MHz, Deuterated-d6) δ = 8.22-8.27 (d, J = 7.1 Hz, 1H), 6.82-6.43 (m, 1H), 5.44-5. 19 (m, 1H), 4.64-4.40 (m, 1H), 3.68 (s, 3H), 1.42 (s, 9H), 1.38-1.40 (d, 3H)
¹⁹ F NMR (376MHz, Deuterated-d6) δ = -80.91 (t, J = 10.0Hz, 3F), -113.87 to -115.77 (m, 1F), 119.22 to 119 .98 (m, 1F), -120.67 to -121.35 (m, 2F), -121.48 (m, 6F), -122.03 to -122.81 (m, 2F), -126 .00 (m, 2F)

[Example 11]
A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Leu-OMe) was synthesized.

2-((tert-butoxycarbonyl) amino) -3,3, in the same manner as in Example 9 except that leucine methyl ester hydrochloride was used instead of glycine methyl ester hydrochloride and silica gel chromatography was not performed. From 4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorododecanoic acid (15 μmol) to Boc-RFAA (C8) -Leu-OMe ( A 49:51 mixture of diastereomers A and diastereomers B) was obtained (83% yield).

^{1 1} H NMR (400 MHz, Deuterated-d6) δ = 8.19 (d, J = 7.5 Hz, 1H), 6.64 (d, J = 10.1 Hz, 1H), 5.45-5.13 ( m, 1H), 4.68-4.47 (m, 1H), 3.68 (s, 3H), 1.81-1.66 (m, 1H), 1.42 (s, 9H), 0 .96-0.88 (m, 6H)
¹⁹ F NMR (376 MHz, Deuterated-d6) δ = -81.56 (t, J = 10.0 Hz, 3F), -115.49 (m, J = 272.51F), 119.40 to -120. 88 (m, 1F), -121.33 to -123.04 (m, 10F), -126.63 (m, 2F)

[Example 12]
A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Lys (Boc) -OMe) was synthesized.

Boc-RFAA (C8) -Lys (Boc) -OMe was obtained in the same manner as in Example 9 except that lysine (Boc) methyl ester hydrochloride was used instead of glycine methyl ester hydrochloride (yield 69%). ).

^{1 1} H NMR (400 MHz, CDCl ₃ ) δ 7.71 (d, J = 3.7 Hz, 1H), 7.51-7.54 (m, 1H), 6.94 (d, J = 8.2 Hz, 1H), 5.67 (d, J = 10.1Hz, 1H), 5.13 (s, 1H), 4.57-4.62 (m, 2H), 3.75 (s, 3H), 1 .45 (s, 18H), 1.10-1.90 (m, 6H)
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ-80.63 (s, 3F), -114.56 (d, J = 281.1 Hz, 1F), 119.10 (d, J = 284.0 Hz, 1F) ), -122.61-120.86 (m, 10F), -126.02 (s, 2F)

LRMS (ESI-TOF)
[M + Na] ⁺ : calcd. for C ₂₇ H ₃₄ F ₁₇ N ₃ NaO ₇ 858.20, found 858.03

[Example 13]
A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Cys (Bn) -OMe) was synthesized.

2-((tert-butoxycarbonyl) amino) -3,3,4 in the same manner as in Example 9 except that S-benzyl-L-cysteine methyl ester hydrochloride was used instead of phenylalanine methyl ester hydrochloride. From 4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorododecanoic acid (36.4 μmol), Boc-RFAA (C8) -Cys (Bn) ) -OMe (diastereomer A: 10.6 μmol, diastereomer B: 9.4 μmol, total yield 55%) was obtained.

Boc-RFAA (C8) -Cys (Bn) -OMe diastereomer A
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.39 (br, 1H), 7.35-7.24 (m, 5H), 6.75 (br, 1H), 5.44-5.38 (M, 1H), 4.81-4.77 (m, 1H), 3.80 (s, 2H), 3.71 (s, 3H), 2.96-2.82 (m, 2H), 1.41 (s, 9H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), -114.59 (d, J = 288Hz, 1F), 119.49 (d, J = 277Hz, 1F), -120.70 to -122.50 (m, 10F), -126.01 (s, 2F)

Boc-RFAA (C8) -Cys (Bn) -OMe diastereomer B
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.40 (br, 1H), 7.36-7.24 (m, 5H), 6.74 (br, 1H), 5.42-5.35 (M, 1H), 4.76-4.72 (m, 1H), 3.80 (s, 2H), 3.70 (s, 3H), 2.98-2.82 (m, 2H), 1.42 (s, 9H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), -114.44 (d, J = 279Hz, 1F), -119.51 (d, J = 271Hz, 1F), -120.65 to -122.65 (m, 10F), -125.96 (s, 2F)

[Example 14]
A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Asp (OtBu) -OMe) was synthesized.

2-((tert-butoxycarbonyl) amino) -3,3 in the same manner as in Example 9 except that Ot-butyl-L-aspartic acid methyl ester hydrochloride was used instead of phenylalanine methyl ester hydrochloride. , 4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-from heptadecafluorododecanoic acid (18.7 μmol), Boc-RFAA (C8)- Asp (OtBu) -OMe (a mixture of diastereomers A and diastereomers B) was obtained (total of diastereomers A and diastereomers B was 13.8 μmol, total yield was 74%).

Boc-RFAA (C8) -Asp (OtBu) -OMe diastereomer A
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.30-8.27 (m, 1H), 6.74-6.72 (m, 1H), 5.37-5.30 (m, 1H) , 4.84-4.82 (m, 1H), 3.70 (s, 3H), 2.97-2.82 (m, 2H), 1.41 (s, 18H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), -114.55 (d, J = 273Hz, 1F), 119.39 (d, J = 286Hz, 1F), -120.70 to -122.50 (m, 10F), -125.95 (s, 2F)

Boc-RFAA (C8) -Asp (OtBu) -OMe diastereomer B
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.34 (br, 1H), 6.68 (br, 1H), 5.45-5.38 (m, 1H), 4.84-4.78 (M, 1H), 3.71 (s, 3H), 2.89-2.76 (m, 2H), 1.43 (s, 9H), 1.41 (s, 9H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), 114.57 (d, J = 275Hz, 1F), 119.35 (d, J = 275Hz, 1F), -120.61 to -122.60 (m, 10F), -125.97 (s, 2F)

[Example 15]
A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Tyr (OBn) -OMe) was synthesized.

2-((tert-butoxycarbonyl) amino) -3,3,4 in the same manner as in Example 9 except that O-benzyl-L-tyrosine methyl ester hydrochloride was used instead of phenylalanine methyl ester hydrochloride. From 4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorododecanoic acid (17.7 μmol), Boc-RFAA (C8) -Tyr (OBn) ) -OMe (diastereomer A: 2.9 μmol, diastereomer B: 2.9 μmol, total yield 32%) was obtained.

Boc-RFAA (C8) -Tyr (OBn) -OMe diastereomer A
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.21 (br, 1H), 7.47-7.32 (m, 5H), 7.02 (dd, 4H), 6.63 (br, 1H) ), 5.38-5.27 (m, 1H), 5.08 (s, 2H), 4.75-4.70 (m, 1H), 3.67 (s, 3H), 3.12- 2.94 (m, 2H), 1.43 (s, 9H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), -114.65 (d, J = 284Hz, 1F), -119.33 (d, J = 273Hz, 1F), -120.30 to -122.60 (m, 10F), -125.96 (s, 2F)

Boc-RFAA (C8) -Tyr (OBn) -OMe diastereomer B
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.23 (br, 1H), 7.47-7.30 (m, 5H), 7.04 (dd, 4H), 6.65 (br, 1H) ), 5.34-5.25 (m, 1H), 5.08 (s, 2H), 4.72-4.68 (m, 1H), 3.66 (s, 3H), 3.10- 2.97 (m, 2H), 1.42 (s, 9H)
¹⁹ F NMR (470 MHz, ACETONE-D6) δ = -80.86 (t, 3F), -114.66 (d, J = 284 Hz, 1F), 119.30 (d, J = 281 Hz, 1F), -120.00 to -122.60 (m, 10F), -125.95 (s, 2F)

[Example 16]
A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Glu (OBn) -OMe) was synthesized.

2-((tert-butoxycarbonyl) amino) -3,3,4 in the same manner as in Example 9 except that O-benzyl-L-glutamic acid methyl ester hydrochloride was used instead of phenylalanine methyl ester hydrochloride. From 4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorododecanoic acid (35.2 μmol), Boc-RFAA (C8) -Glu (OBn) ) -OMe (a mixture of diastereomers A and diastereomers B) was obtained (total of diastereomers A and diastereomers B was 27.1 μmol, total yield was 76.9%).

Boc-RFAA (C8) -Glu (OBn) -OMe diastereomer A
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.24 (br, 1H), 7.38-7.30 (m, 5H), 6.74 (br, 1H), 5.32-5.26 (M, 1H), 5.11 (s, 2H), 4.62-4.58 (m, 1H), 3.69 (s, 3H), 2.51-2.48 (m, 2H), 3.69 (s, 3H), 2.28-1.90 (m, 2H), 1.41 (s, 9H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), -114.50 (d, J = 293Hz, 1F), -119.43 (d, J = 295Hz, 1F), -120.05 to -122.70 (m, 10F), -125.93 (s, 2F)

Boc-RFAA (C8) -Glu (OBn) -OMe diastereomer B
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.33 (br, 1H), 7.39-7.30 (m, 5H), 6.70 (br, 1H), 5.37-5.29 (M, 1H), 5.11 (s, 2H), 4.61-4.57 (m, 1H), 3.70 (s, 3H), 2.62-2.51 (m, 2H), 2.25-1.95 (m, 2H), 1.40 (s, 9H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), -114.80 (d, J = 273Hz, 1F), -119.62 (d, J = 279Hz, 1F), -120.05 to -122.50 (m, 10F), -125.97 (s, 2F)

[Example 17]
A dipeptide having a heptadecafluorooctyl group (Boc-RFAA (C8) -Arg (Pbf) -OMe) was synthesized.

2-((tert-butoxycarbonyl) amino) -3,3,4 in the same manner as in Example 9 except that N-Pbf-L-arginine methyl ester hydrochloride was used instead of phenylalanine methyl ester hydrochloride. From 4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorododecanoic acid (17.3 μmol), Boc-RFAA (C8) -Arg (Pbf) )-OMe (mixture of diastereomer A and diastereomer B) was obtained (total of diastereomer A and diastereomer B 13.2 μmol, total yield 76.3%).

Boc- [RFAA (C8) -Arg (Pbf) -OMe diastereomer A and diastereomer B mixture
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.35, 8.27 (m, 1H (NH)), 6.76-6.72 (m, 1H (NH)), 6.60-6. 30 (br, 3H (NH)), 4.50-4.45 (m, 1H), 3.67 (s, 3H), 3.30-3.15 (m, 2H), 2.99 (s) , 2H), 2,56, 2.49 (s2, 9H), 1.80-1.50 (m, 2H), 1.44 (s, 6H), 1.41 (s, 9H), 1 .45-1.38 (m, 2H)
¹⁹ F NMR (470 MHz, ACETONE-D6) δ = -80.86 (t, 3F), 114.20-114.98 (m, 1F), 119.00-120.20 (m, 1H), -120.72 to -122.52 (m, 10F), -125.98 (s, 2F)

[Comparative Example 4]
A tripeptide having an octyl group (Boc-Ala-nOctyl-Phe-OMe) was synthesized.

Boc-nOctyl-Phe-OMe was manufactured by the method described in the previous report (Liebigs Annalen der Chemie, 1990, 12p, p. 1175-1183). Boc deprotection of the dipeptide was performed by the same standard procedure as above (yield 100%). In addition, the synthesis of tripeptide was carried out according to the above-mentioned procedure.

ESI-MS
[M + H] ⁺ : m / z calcd. for 420.29, found 420.72

[Manufacturing Example 4]
From 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyliodide and compound 1 below, 2-amino-5,5,6,6 7,7,8,8,9,9,10,10,10-tridecafluorooctanoic acid (hydrochloride) was synthesized.

Compound 1 (200 mg) dissolved in THF (1 mL) was added to LDA (2.2 eq) and the resulting mixture was kept at −78 ° C. in dry THF (1 mL) for 30 minutes under argon. RFCH ₂ CH ₂ I (1.1 eq) was then added to the mixture and stirred for 3 hours. Then, the mixture was slowly brought to −30 ° C., stirred overnight, and then water (5 mL) was added to the mixture at 0 ° C. to stop the reaction. The mixture was then extracted with CH ₂ Cl ₂ (200 mL x 3). The product was cast on alumina to give a white solid (yield 54.1%).

^{1 1} H NMR (400MHz CDCl ₃ ) δ = 7.2-7.7 (m, 10H), 4.03 (t, 1H), 3.23-3.27 (m, 2H), 2.78-2 .66 (m, 2H), 1.45 (s, 9H)
¹⁹ F NMR (376MHz CDCl ₃ ) δ = -80.8 (t, 3F), -114.2 (m, 1F), -114.9 (m, 1F), -121.9 (m, 2F), -122.9 (m, 2F), -123.4 (m, 2F), -126.2 (m, 2F)

A solution of compound 2 (50.8 mmol) dissolved in HCl (6M, 50 mL) and 1,4-dioxane (200 mL) was heated at 80 ° C. for 24 hours. The solution was filtered and the precipitate was washed with acetone several times. The resulting white solid was of sufficient purity for use in the next step without further purification (yield 50.2%).

^{1 1} H NMR (400MHz MeOH-d4) δ = 3.68 (m, 1H), 2.52 (m, 1H), 2.35 (m, 1H), 2.11 (m, 2H)
¹⁹ F NMR (376MHz MeOH-d4) δ = -82.3 (t, 3F), -115.7 (m, 2F), -122.8 (m, 2F), -123.8 (m, 2F) , -124.4 (m, 2F), -127.2 (m, 2F)

[Example 18]
A tripeptide having a tridecafluorohexyl group (H-Ala-RFAA (C6) -Phe-OMe) was synthesized.

A stir bar was added to a 50 mL two-necked round bottom flask, and compound 3 (100 mg, 0.22 mmol) and DCM (10 mL) were added. Fmoc-OSn (0.24 mmol) and DIPEA (1.3 eq) were added at room temperature, then the reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was directly evaporated and purified by silica gel column chromatography (MeOH / CHCl ₃ = 1/9) as a white solid (64.2% yield).

Fmoc-RFAA (C6) -OH (90.4 mg, 0.14 mmol), HOAt (1.2 eq), and DIPEA (1.3 eq) were added to a 25 mL two-necked round bottom flask. HATU (1.2 eq) and H-Phe-OMe (HCl salt, 1.2 eq) dissolved in DCM (5 mL) were added to the mixture at 0 ° C. to warm the mixture to room temperature, then 4 Stir for hours. The reaction was then stopped by adding HCl (1N) and then the mixture was partitioned between HCl (1N) and DCM. The combined organic phases were evaporated and then diluted with ethyl acetate. The organic phase was washed with HCl (1N), saturated aqueous NaHCO ₃ solution, and brine, dried over v and evaporated to give a white solid. A 20% piperidine DMF solution (25 mL) was added to the crude mixture, and the mixture was stirred at room temperature for 1 hour. The solvent was removed by vacuum drying to give a white solid, which was purified by silica gel column chromatography (CHCl ₃ : MeOH = 10: 1) and H-RFAA (C6) -Phe-OMe (41.4 mg, yield). 64.8%) was obtained.

^{1 1} H NMR (400MHz Deuterated-d6) δ = 7.2-7.7 (m, 5H), 4.71 (m, 1H), 4.05 (t, 1H), 3.15 (s, 3H) , 2.99-3.15 (m, 2H), 2.81 (br s, NH ₂ ), 2.32 (m, 2H), 1.96 (m, 2H)
¹⁹ F NMR (376MHz Deuterated-d6) δ = -81.4 (t, 3F), -114.6 (m, 2F), -122.4 (m, 2F), -123.4 (m, 2F) , -123.8 (m, 2F), -126.7 (m, 2F)

H-RFAA (C6) -Phe-OMe (44.8 μmol), HOAt (1.2 eq), and DIPEA (1.3 eq) were added to a 25 mL two-necked round bottom flask. HATU (1.2 eq) and Fmoc-AlaOH (1.2 eq) dissolved in DCM (5 mL) were added to the mixture at 0 ° C., the mixture was warmed to room temperature and then stirred for 4 hours. After stopping the reaction by adding HCl (1N), the mixture was partitioned between HCl (1N) and DCM. The combined organic phases were evaporated and then diluted with ethyl acetate. The organic phase was washed with HCl (1N), saturated aqueous NaHCO ₃ solution, and brine, dried over Na ₂ SO ₄ and evaporated to give a white solid. 5 mL of a 20% piperidine DMF solution was added to the crude mixture, and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure to obtain a white solid, which was purified by HPLC.

ESI-MS
[M + H] ⁺ : m / z calcd. for 754.16, found 654.52

[Example 19]
A dipeptide having a tridecafluorooctyl group (Fmoc-RFAA (C6) -Phe-OMe) was synthesized.

2-((tert-butoxycarbonyl) amino) -3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorododecane 2-((9-Fluorenylmethyloxycarbonyl) amino) -5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorododecane instead of acid Fmoc-[(R)- RFAA (C6)]-Phe-OMe (diastereomer A) (110.9 μmol) and Fmoc-[(S) -RFAA (C6)]-Phe-OMe (diastereomer B) (137.3 μmol) are obtained. (Total yield 78.9%).

Here, using the crystal of diastereomer B, the solid of RFAA (C6) contained by X-ray structural analysis was determined to be the (S) form. Rigaku's VariMax Dual Saturn was used for the X-ray structure analysis.

Fmoc-[(R) -RFAA (C6)]-Phe-OMe (Diastereomer A)
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.03 (t, 2H), 7.87 (d, 2H) 7.72-7.68 (m, 2H), 7.42 (t, 2H) , 7.32 (t, 2H), 7.28-7.14 (m, 5H), 4.74-4.68 (m, 1H), 4.40-4.35 (m, 2H), 4 .36-4.28 (m, 1H), 4.28-4.20 (m, 1H), 3.66 (s, 3H), 3.20-3.00 (m, 2H), 2.40 -2.00 (m, 4H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (s, 3F), -114.00 (s, 2F), -121.66 (s, 2F), -122.64 (s, 2F) ), -123.13, -126.00 (s, 2F)

Fmoc-[(S) -RFAA (C6)]-Phe-OMe (Diastereomer B)
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.03 (s, 2H), 7.87 (d, 2H) 7.72-7.66 (m, 2H), 7.41 (t, 2H) , 7.31 (t, 2H), 7.26-7.16 (m, 5H), 4.75-4.70 (m, 1H), 4.40-4.28 (m, 3H), 4 .25-4.20 (m, 1H), 3.67 (s, 3H), 3.23-2.95 (m, 2H), 2.33-1.79 (m, 4H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), -113.89 (s, 2F), -121.67 (s, 2F), -122.64 (s, 2F) ), -123.02, 126.00 (s, 2F)

The protecting group on the N-terminal side of the synthesized peptide was deprotected to obtain H-[(R) -RFAA (C6)]-Phe-OMe.

Place the stir bar in a 5 mL test tube, and add Fmoc-[(R) -RFAA (C6)]-Phe-OMe (diastereomer A, 0.111 mmol), dimethylformamide (2.6 mL), and piperidine. 0.6 mL of the mixed solution was added. The reaction mixture was stirred at room temperature for 1 hour, then low boiling and the solvent was distilled off under reduced pressure. Hexane was added to the residue, washed, and the insoluble solid was filtered. Then, the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography (chloroform / methanol = 10/1) to give H-[(R) -RFAA (C6)]-Phe-OMe (diastereomer A) (0.053 mmol). , Yield 47.4%).

H-[(R) -RFAA (C6)]-Phe-OMe (Diastereomer A)
^{1 1} H NMR (500MHz, ACETONE-D6) δ = 7.90 (br, 1H), 7.50 (br, 1H), 7.30-7.20 (m, 5H), 4.76-4.71 (M, 1H), 3.69 (d, 3H), 3.40-3.29 (m, 1H), 3.22-3.04 (m, 2H), 2.40-1.66 (m) , 4H)
¹⁹ F NMR (470MHz, ACETONE-D6) δ = -80.86 (t, 3F), -113.90 (s, 2F), -121.69 (s, 2F), -122.65 (s, 2F) ), -123.15 (s, 2F), -125.98 (s, 2F)

The fluorescent substance AlexaFluor 647 was bound to the N-terminal of the synthesized peptide after deprotection (H-[(R) -RFAA (C6)]-Phe-OMe, diastereomer A).

Alexa Fluor 647 NHS ester (500 μg) dissolved in dry DMSO (50 μL) and H-[(R) -RFAA (C6)] -Phe dissolved in dry DMSO (50 μL) in a 1.5 mL black tube. -OMe (3.0 eq) and DIPEA (3.0 eq) dissolved in dry DMSO (20 μL) were added. The mixture was continuously stirred at room temperature overnight. The mixture was purified by reverse phase chromatography (acetonitrile / water / TFA = 25: 75: 0.1 to 99: 1: 0.1) and lyophilized to give the fluorescent conjugate 3 as a blue solid (fluorescence). Yield 53.4% calculated by total (based on dye). The fluorescence was measured at an emission wavelength of 650 nm using a NanoDrop (registered trademark) spectrophotometer ND-1000.

MALDI-TOF MS
[M + 2] ^- : m / z calcd for C ₅₆ H ₆₁ F ₁₃ N ₄ O ₁₆ S _4-1421.3369 , found 1421.4965

[Manufacturing Example 5]
From 1,1,2,2,3,3,4,5,5,6,6,6-tridecafluorohexyl iodide and ethyl propiolate, phthalimide, 2- (1,3-dioxoiso) Indoline-2-yl) -3-perfluorohexylpropanoic acid was synthesized.

Synthesized by the reaction described below according to the previous report (Chemical Communications, 2015, vol.51, p.12451, and Journal of Fluorine Chemistry, 2013, vol.150, p.1-7).

[Example 20]
A dipeptide having a tridecafluorohexyl group (Phth-RFAA (C6') -Phe-OMe) was synthesized.

L-L-phenylalanine methyl ester hydrochloride 53.7 mg (0.10 mmol), oxyma 14.2 mg (0.10 mmol), COMU 42.8 mg (0.10 mmol) in a 10 mL two-necked round-bottom flask. , 2- (1,3-Dioxoisoindoline-2-yl) -3-perfluorohexyl propanoic acid (53.7 mg, 0.10 mmol) was added and dissolved in 3.0 mL of dehydrated dimethylformamide. Subsequently, 25.8 mg (0.20 mmol) of diisopropylamine was added dropwise to the mixture, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was diluted with 20 mL of ethyl acetate, treated with 20 mL of 1N hydrochloric acid and 20 mL of saturated aqueous sodium hydrogen carbonate solution, separated, and then the organic layer was washed with saturated aqueous sodium chloride solution. The obtained organic layer was dehydrated with sodium sulfate, and the solvent was distilled off. The obtained crude product was purified by HPLC fractionation to obtain Phth-RFAA (C6') -Phe-OMe (23 mg, 0.030 mmol, yield 33%).

^{1 1} HNMR (400MHz, CDCl ₃ ) δ 7.90-7.87 (m, 2H), 7.82-7.77 (m, 2H), 7.27-7.10, 7.00-6.93 (M, 5H), 6.40, 6.20 (d, J = 7.8Hz, 1H), 5.22 (td, J = 7.0, 3.7Hz, 1H), 4.87-4. 79 (m, 1H), 3.72, 3.68 (s, 3H), 3.25-3.00 (m, 4H)
¹⁹ F NMR (376 MHz, CDCl ₃ ) δ-80.67 (s, 3F), -114.80 (brs, 2F), -121.71 (s, 2F), -122.77 (s, 2F), -123.40 (s, 2F), -126.06 (s, 2F)

[Example 21]
A tetrapeptide having a heptadecafluorooctyl group (Boc-Cys (Trt) -Ala-RFAA (C8) -Phe-OMe) was synthesized.

Tripeptide (H-Ala-[(R) -RFAA (C8)]-Phe-OMe (diastereomer A, 27.6 μmol), Boc-Cys (Trt) -OH in a 25 mL two-necked round bottom flask. (1.8 eq), flask (1.8 eq), and COMU (1.8 eq) were dissolved in 600 μL DCM and stirred. The reaction mixture was cooled to 0 ° C. and DIPEA (1.8 eq) was added. After the addition, the temperature was raised to room temperature and the mixture was stirred for 3.5 hours. Then, the reaction mixture was quenched with HCl (1N) and extracted 3 times with DCM. The combined organic phases were concentrated under reduced pressure and then with ethyl acetate. It was diluted and washed with HCl (1N), saturated aqueous sodium hydrogen carbonate, and saturated brine. The washed organic phase was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to Boc-Cys (Trt) -Ala-RFAA. A crude product of (C8) -Phe-OMe was obtained. Subsequently, the crude product was purified by silica gel chromatography (chloroform / methanol = 10/1) to obtain Boc-Cys (Trt) -Ala-[(R). ) -RFAA (C8)]-Phe-OMe (diastereomer A) was obtained (20.6 μmol, yield 74.6%).

Boc-Cys (Trt) -Ala-[(R) -RFAA (C8)]-Phe-OMe (Diastereomer A)
^{1 1} H NMR (500MHz, ACETONE-D6) δ = 8.18, 7.86 (br, 2H), 7.43-7.14 (m, 20H), 6.09 (br, 1H), 5.65 -5.57 (m, 1H), 4.77 (t, 1H), 4.50-4.43 (m, 1H), 4.00-3.92 (m, 1H), 3.68 (s) , 3H), 3.20-3.00 (m, 2H), 2.65-2.57 (m, 2H) 1.41 (s, 9H), 1.26 (d, 3H)
¹⁹ F NMR (470 MHz, ACETONE-D6) δ = -80.86 (t, 3F), -114.51 (d, J = 289 Hz, 1F), -118.48 (d, J = 293 Hz, 1F), -120.00 to -122.60 (m, 10F), -125.88 (s, 2F)

The protecting group on the N-terminal side of the synthesized peptide was deprotected to obtain H-Cys (Trt) -Ala-RFAA (C8) -Phe-OMe.

Place the stir bar in a 25 mL two-necked flask and place Boc-Cys (Trt) -Ala-[(R) -RFAA (C8)] -Phe-OMe (diastereomer A, 20.2 μmol) and DCM (3 mL). Was added. After cooling this reaction mixture to 0 ° C., a solution of triisopropylsilane (53 μL) and TFA (1 mL) in 3 mL of DCM was added, and the temperature was raised to room temperature. After stirring for 3 hours, an aqueous sodium hydrogen carbonate solution was added to terminate the reaction. The aqueous phase was extracted with DCM and the combined organic phase was distilled off under reduced pressure to give a crude product of the deprotected H-Cys-Ala-RFAA (C8) -Phe-OMe. The crude product was purified by silica gel chromatography (chloroform / methanol = 20/1) to obtain H-Cys-Ala-[(R) -RFAA (C8)]-Phe-OMe (diastereomer A). (13.4 μmol, yield 66.5%).

H-Cys-Ala-[(R) -RFAA (C8)]-Phe-OMe (Diastereomer A)
^{1 1} H NMR (500 MHz, ACETONE-D6) δ = 8.34 (d, 1H), 7.98-7.93 (br, 2H), 7.28-7.18 (m, 5H), 5.68 -5.60 (m, 1H), 4.80-4.75 (m, 1H), 4.61-4.55 (m, 1H), 4.30-4.22 (m, 1H), 3 .68 (s, 3H), 3.36-3.15 (m, 2H), 1.35 (d, 3H)
¹⁹ F NMR (470 MHz, ACETONE-D6) δ = -80.86 (s, 3F), 114.58 (d, J = 286 Hz, 1F), -118.99 (d, J = 281 Hz, 1F), -120.00 to -122.46 (m, 10F), -125.89 (s, 2F)

The fluorescent substance AlexaFluor 647 was bound to the terminal SH group of the synthesized peptide after deprotection (H-Cys-Ala-RFAA (C8) -Phe-OMe).

AlexaFluor 647 C2 maleimide (500 μg) dissolved in PBS (50 μL of phosphate saline) and H-Cys-Ala-RFAA (C8) dissolved in dried DMSO (50 μL) in a 1.5 mL black tube. -Phe-OMe (3.0 equivalents), dried DMSO (20 μL) was added. The mixture was continuously stirred at room temperature overnight. The mixture was purified by reverse phase chromatography (acetonitrile / water / TFA = 25: 75: 0.1 to 99: 1: 0.1) and lyophilized to give the fluorescent conjugate 4 as a blue solid (fluorescence). Yield 18.6% calculated by total (based on dye). The fluorescence was measured at a emission wavelength of 650 nm using a NanoDrop (registered trademark) spectrophotometer ND-1000.

MALDI-TOF MS
[M + 3] ^- : m / z calcd for C ₆₈ H ₇₇ F ₁₇ N ₈ O ₂₀ S _5-1809.6745 , found1809.6536

[Test Example 1]
Peptide fluorescent conjugate 1 (Alexa-Ala-[(R) -RFAA (C8)]-Phe-OMe, diastereomeric A) and peptide fluorescent conjugate 2 (Alexa-Ala-[(S)) synthesized in Example 8 ) -RFAA (C8)]-Phe-OMe, Diasteromer B), Peptide Fluorescence Conjugate 3 (Exa-[(R) -RFAA (C6)]-Phe-OMe) synthesized in Example 19, Example. Peptide fluorescence conjugate 5 synthesized in 6 (Alexa-Ala-[(R) -RFAA (C4)]-Phe-OMe), peptide fluorescence conjugate 6 synthesized in Comparative Example 3 (Alexa-Ala-Nle-Phe-) The efficiency of intracellular uptake of OMe) and the diethylamide (fluorescent dye 1) of the fluorescent substance Alexa Fluor 647 was investigated.

(Preparation of sample solution)
The DMSO solutions of the synthesized peptide fluorescent conjugates 1, 2, 3, 5, 6 and fluorescent dye 1 for 37 ° C. DMEM medium conditions were diluted with DMEM low glucose culture medium to a concentration of 1.5 μM. This was used as a sample solution for testing at 37 ° C. Since each sample solution contains 0.15% DMSO, DMEM low glucose liquid medium containing 0.15% DMSO was used for blank measurement.

-For 4 ° C buffer conditions A DMSO solution of the synthesized peptide fluorescent conjugates 1, 2, 3 and fluorescent dye 1 diluted with 25 mM HEPES buffer to a concentration of 1.5 μM was diluted at 4 ° C. It was used as a test sample solution. For blank measurements, 25 mM HEPES buffer containing 0.15% DMSO was used.

(Evaluation of cell membrane permeability)
-Changes over time in DMEM medium conditions at 37 ° C. 1. Comparison of Peptide Fluorescent Conjugates 1, 2, 5, 6 and Fluorescent Dye 1 A medium of HeLa cells precultured at 37 ° C. for 24 hours was used as a sample solution of peptide fluorescent conjugates 1, 2, 5, 6 and fluorescent dye 1. After substitution and culturing at 37 ° C. for 1 hour, 4 hours, and 24 hours, the cell membrane permeability was evaluated. After culturing for each predetermined time, the cell surface was washed 3 times with PBS (phosphate saline), and the cells were peeled off and collected with Trypin-EDTA 0.05% (manufactured by Gibco). The recovered cells were analyzed by measuring and analyzing red 2 fluorescence (661 / 15 nm) for detecting the fluorescent dye (Alexa Fluor 647) introduced into the peptide fluorescence conjugate by flow cytometry (guava easeCyte ™ 8). .. The results of flow cytometric analysis of cells cultured at 37 ° C. for 1 hour in the sample solution are shown in FIG. The vertical axis is the number of cells (count), and the horizontal axis is the fluorescence intensity of each cell. The results of comparing the average fluorescence intensities of each sample are shown in FIG. Further, FIG. 3 shows a comparison of average fluorescence intensities in cells cultured at 37 ° C. for 1 hour, 4 hours, and 24 hours. In each figure, the peptide fluorescent conjugate is referred to as PFCJ, and the fluorescent dye is referred to as FD.

2. 2. Comparison of peptide fluorescence conjugates 1, 2 and 3 Red 2 fluorescence (661 / 15 nm) was measured by flow cytometry in the same manner as above except that the sample solutions of peptide fluorescence conjugates 1, 2 and 3 were used. analyzed.
The analysis results by flow cytometry of the cells cultured at 37 ° C. for 1 hour and 4 hours in the sample solution are shown in FIGS. 4 and 5. In each figure, the peptide fluorescent conjugate is shown as PFCJ, and the fluorescent dye is shown as FD.

・ 4 ° C buffer condition The medium of HeLa cells pre-cultured at 37 ° C for 24 hours was replaced with 25 mM HEPES buffer previously cooled to 4 ° C, cultured at 4 ° C for 1 hour, and then further tested at 4 ° C. It was replaced with a sample solution and cultured at 4 ° C. for 40 minutes. The cultured cells were analyzed by flow cytometry in the same procedure as that performed under the condition of 37 ° C. under the condition that the temperature of the sample did not rise. The results are shown in FIG.

As shown in FIG. 1, regarding the fluorescence intensity of AlexaFluor 647 of cells after incubation at 37 ° C. for 1 hour, cells treated with a peptide fluorescent conjugate 6 containing no fluorine atom in the side chain were treated with fluorescent dye 1. The cells treated with the peptide fluorescent conjugates 1, 2 and 5 containing a fluorine atom in the side chain were all treated with the fluorescent dye 1 or the peptide fluorescent conjugate 6 while the cells were treated with the fluorescent dye 1 or the peptide fluorescent conjugate 6. It was higher than the cells. As shown in FIG. 2, the average value of the fluorescence intensity of Alexa Fluor 647 of the cells is about 9 for the cells treated with the peptide fluorescence conjugate 1 as compared with the cells treated with the peptide fluorescence conjugate 6 or the fluorescent dye 1. The cells treated with the peptide fluorescence conjugate 2 were more than twice as high, and the cells treated with the peptide fluorescence conjugate 5 were more than twice as high. From these results, it was found that peptides having a fluoroalkyl group in the side chain have higher intracellular uptake efficiency and excellent cell membrane permeability as compared with peptides and fluorescent dyes having no fluoroalkyl group. ..

In particular, peptide fluorescent conjugates 1 and 2 having a large number of carbon atoms and fluorine atoms in the side chain have higher cell membrane permeability than peptide fluorescent conjugate 5 having a relatively small number of carbon atoms and fluorine atoms in the side chain, and are cells. Was taken in efficiently. The average value of the fluorescence intensity of Alexa Fluor 647 of the cells was about 4 times that of the cells treated with the peptide fluorescence conjugate 5 as compared with the cells treated with the peptide fluorescence conjugate 5, and the cells treated with the peptide fluorescence conjugate 2 were treated with the peptide fluorescence conjugate 2. It was about 14 times higher in the cells. That is, the peptide fluorescent conjugates 1 and 2 having a heptadecafluorooctyl group were superior in cell membrane permeability to the peptide fluorescent conjugate 5 having a nonafluorobutyl group.
Furthermore, by comparing the results of peptide fluorescence conjugates 1 and 2, it was also found that the cell membrane permeability changes depending on the configuration of the asymmetric points present in the fluorine-containing amino acid (RFAA (C8)).

Further, also in the result of FIG. 3, similarly to the result of FIG. 2, the average value of the fluorescence intensity of AlexaFluor 647 of the cells treated with the peptide fluorescence conjugates 1, 2 and 5 containing a fluorine atom in the side chain is the side chain. It was higher than the cells treated with the peptide fluorescent conjugate 6 or the fluorescent dye 1 containing no fluorine atom. From the results shown in FIG. 3, the proportion of cells that emit fluorescence increased over time in the cells treated with any of the peptide fluorescent conjugates, and the amount of peptide fluorescent conjugates taken up by the cells under the test conditions of 37 ° C. Was also confirmed to increase over time.

As shown in FIG. 4, the peptide fluorescent conjugate 3 having the same side chain carbon number but a small number of fluorine atoms has a cell membrane permeability equal to or higher than that of the peptide fluorescent conjugate 1 after 1 hour at 37 ° C. As shown in FIG. 5, after 4 hours, the cell membrane permeability was close to that of the peptide fluorescent conjugate 2. From this, it was found that by adjusting the number of carbon atoms in the side chain, the same or higher cell membrane permeability can be obtained even if the number of fluorine atoms is reduced.

As shown in FIG. 6, even in the cells after incubation at 4 ° C. for 40 minutes, the fluorescence intensity and the average value of AlexaFluor 647 of the cells treated with peptide fluorescent conjugates 1 to 3 containing a fluorine atom in the side chain are shown. , It was clearly higher than the cells treated with the fluorescent dye 1, and it was confirmed that these peptides were efficiently taken up into the cells even at 4 ° C. Since active transport from the outside of the cell to the inside of the cell is stopped and only passive transport is performed under the condition of 4 ° C., the peptide having a fluoroalkyl group is excellent in that it enhances cell permeability in both passive transport and active transport. It turned out to have the property.

[Test Example 2]
The peptide fluorescent conjugate 4 (Alexa-Cys-Ala-RFAA (C8) -Phe-OMe) and the peptide fluorescent conjugate 1 (Alexa-Ala-[(R) -RFAA (C8)]-Phe synthesized in Example 21). -OMe, diastereomer A) and the diethylamide form (fluorescent dye 1) of the fluorescent substance Alexa Fluoro 647 were examined for their uptake efficiency into cells. The sample solution was prepared in the same manner as in Test Example 1, and the cells cultured at 37 ° C. for 1 hour, 4 hours, and 24 hours were analyzed by flow cytometry, and the average fluorescence intensity was compared.

The average fluorescence intensity of Alexa Fluor 647 of cells after incubation at 37 ° C. for 1 hour, 4 hours, and 24 hours was a fluorescent dye in cells treated with peptide fluorescent conjugates 1 and 4 containing a fluorine atom in the side chain. It was higher than 1 treated cells. Further, in comparison between the peptide fluorescence conjugate 4 derived from the tetrapeptide having the same fluorine-containing alkyl group and the peptide fluorescence conjugate 1 derived from the tripeptide, the peptide fluorescence conjugate was obtained at any of 1 hour, 4 hours and 24 hours. Gate 4 had a higher average fluorescence intensity. From this, it was found that in peptides having a fluorine-containing alkyl group having the same number of carbon atoms and fluorine atoms in the side chain, the cell membrane permeability tends to improve as the number of bound amino acids increases.

[Example 22]
A tripeptide having a tridecafluorohexyl group (H-Ala-[(R) -RFAA (C6)]-Phe-OMe) was synthesized.

Using H-[(R) -RFAA (C6)]-Phe-OMe (diastereomer A, 23 mg) synthesized in Example 19, it was condensed with Boc-Ala-OH in the same manner as in Example 8. By deprotecting the protecting group on the N-terminal side, the same H-Ala-[(R) -RFAA (C6)]-Phe-OMe (9 mg, 41% yield) as that obtained in Example 18 was obtained. (2step yield).

Boc-Ala-[(R) -RFAA (C6)]-Phe-OMe (Diastereomer A)
^{1 1} H NMR (500 MHz, CHLOROFORM-D) δ = 7.30-7.09 (m, 5H), 6.87 (br, 1H), 6.55 (br, 1H) 4.93 (br, 1H) , 4.85 (m, 1H), 4.09 (m, 1H), 3.73 (s, 3H), 3.15-3.09 (m, 2H), 2.18-1.89 (m) , 4H) 1.43 (s, 9H) 1.30 (d, 3H)
¹⁹ F NMR (470 MHz, ACETONE-D6) δ = -80.68 (s, 3F), -114.16 (s, 2F), -121.87 to -123.11 (m, 6F), -126. 06 (s, 2F)

H-Ala-[(R) -RFAA (C6)]-Phe-OMe (Diastereomer A)
1H-NMR (500 MHz, CHLOROFORM-D) 7.07-7.43 (m, 5H), 4.81-4.89 (m, 1H), 4.28 (m, 1H), 3.72 ( s, 3H), 3.41-3.35 (m1H), 3.17 (dd, J = 14.0, 5.4 Hz, 1H), 3.07 (J = 14.0, 5.4 Hz) , 1H), 2.28-2.09 (m, 2H), 2.05-1.93 (m, 2H), 1.32-1.28 (m, 3H)
¹⁹ F NMR (470 MHz, CHLOROFORM-D) δ = -80.68 (s, 3F), -114.31 (s, 2F), -121.58 (s, 2F), -120.40 to -123. 26 (m, 4F), -126.06 (s, 2F)

Subsequently, the fluorescent substance AlexaFluor 647 was bound to the N-terminal of the synthesized deprotected peptide (H-Ala-[(R) -RFAA (C6)]-Phe-OMe, diastereomer A).

Using Alexa Fluor 647 NHS ester (250 μg), the peptide fluorescent conjugate 7 was obtained as a blue solid in the same manner as in Example 8 (calculated by a fluorometer, yield 40% based on the dye).

MALDI-TOF MS
[M + 4] ⁺ : m / z calcd for C ₆₀ H ₇₀ F ₁₃ N ₄ O ₁₇ S ₄ +: 1493.34, found: 1494.28

[Test Example 3]
The peptide fluorescence conjugate 7 (Alexa-Ala-[(R) -RFAA (C6)]-Phe-OMe) synthesized in Example 22 and the peptide fluorescence conjugate 1 (Alexa-Ala- [] synthesized in Example 8). (R) -RFAA (C8)]-Phe-OMe, diastereomers A) and peptide fluorescence conjugate 2 (Alexa-Ala-[(S) -RFAA (C8)]-Phe-OMe, diastereomers B) And the peptide fluorescence conjugate 5 (Alexa-Ala-[(R) -RFAA (C4)]-Phe-OMe) synthesized in Example 6 and the peptide fluorescence conjugate 6 (Alexa-Ala) synthesized in Comparative Example 3. -Nle-Phe-OMe) and the diethylamide form (fluorescent dye 1) of the fluorescent substance Alexa Fluoro 647 were examined for their uptake efficiency into cells.

The sample solution was prepared in the same manner as in Test Example 1, and the analysis results by flow cytometry of cells cultured at 37 ° C. for 1 hour in the sample solution are shown in FIG. In addition, the result of comparing the average fluorescence intensity of each sample is shown in FIG.

The average fluorescence intensity of Alexa Fluor 647 of cells after incubation at 37 ° C. for 1 hour was 1 for fluorescent dye 1 in all cells treated with peptide fluorescent conjugates 1, 2, 5, and 7 containing a fluorine atom in the side chain. It was higher than the cells treated with the peptide fluorescent conjugate 6 containing no fluorine atom in the side chain. In the comparison of peptide fluorescence conjugates 1, 2, and 7 having the same number of carbon atoms in the side chain and containing a fluorine atom in the side chain, the average fluorescence intensity was higher in the order of peptide fluorescence conjugates 2, 7, and 1.

The present invention provides a peptide having an amino acid residue having a fluoroalkyl group in the side chain. Since the peptide according to the present invention has excellent cell membrane permeability, it is expected to be used in the pharmaceutical field as a physiologically active substance such as a carrier for introducing a medicinal ingredient into a target cell.

Claims (5)

1. A peptide in which two or more amino acids are peptide-bonded,
   At least one of the amino acid residues constituting the peptide is C _1-30 alkyl group substituted with at least two fluorine atoms in the side chain, or C _2- replaced with at least two fluorine atoms. A peptide having a group having 1 to 5 ether-bonding oxygen atoms between carbon atoms of a ₃₀ alkyl group.
2. 1 to 5 ether-bonded oxygen atoms between the carbon atoms of the C _1-30 alkyl group substituted with at least two fluorine atoms or the C _2-30 alkyl group substituted with at least two fluorine atoms. The peptide according to claim 1, wherein the group having is further substituted with a halogen atom other than the fluorine atom.
3. 1 to 5 ether-bonded oxygen atoms between the carbon atoms of the C _1-30 alkyl group substituted with at least two fluorine atoms or the C _2-30 alkyl group substituted with at least two fluorine atoms. The side chain having a group having the following general formula (f-1) or (f-2)
   

   

   (In the formula, Rf ^P is between carbon atoms of a fully halogenated C _1-10 alkyl group containing at least two or more fluorine atoms or a fully halogenated C _2-10 alkyl group containing at least two or more fluorine atoms. Represents a group having 1 to 5 ether-bonding oxygen atoms, n1 is an integer of 0 to 10, n2 is an integer of 0 to 9, and a black circle means a bond).
   The peptide according to claim 1 or 2, which is a group represented by.
4. The peptide according to any one of claims 1 to 3, wherein the C-terminal or N-terminal may be protected with a protecting group.
5. The peptide according to any one of claims 1 to 4, which is cell membrane permeable.

Images